Aortic carboxypeptidase-like polypeptide

ABSTRACT

The invention features a aortic carboxypeptidase-like polypeptide (ACLP), DNA encoding ACLP, and methods of detecting genetic alterations associated with abdominal wall defects.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from application U.S. Ser. No.08/818,009 filed on Mar. 14, 1997, which claims priority fromprovisional application U.S. S. No. 60/013,439, filed on Mar. 15, 1996,both of which are hereby incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was funded in part by the U.S. Government undergrant numbers RO1GM awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] The invention relates to gastrointestinal abnormalities.

[0004] Gastroschisis is a life-threatening abdominal wall defect thatoccurs in approximately 1-7 of every 10,000 human births. The defect isthought to originate on the right side of the umbilical cord and mayinvolve the formation of the omphalomesenteric artery. Infants withgastroschisis can be born with abdominal organs outside the body cavity,i.e., protruding through the defect. Factors associated with anincreased risk for gastroschisis include a maternal age below 20 years,ingestion of aspirin, and ingestion of pseudoephedrine. The cause ofgastroschisis has not been identified.

SUMMARY OF THE INVENTION

[0005] A novel human gene encoding aortic carboxypeptidase-likepolypeptide (ACLP) has been discovered. A mutation in an ACLP gene hasnow been shown to be associated with the development of gastroschisis.Thus, a mutation in an ACLP gene is indicative of gastroschisis or apredisposition to develop the condition. Accordingly, the inventionprovides an isolated nucleic acid (e.g., genomic DNA, cDNA, or syntheticDNA) encoding an ACLP. By the term “human ACLP” is meant a polypeptidehaving the amino acid sequence of a naturally-occurring human ACLP. Forexample, the invention encompasses an ACLP with the amino acid sequenceof SEQ ID NO: 2 as well as naturally-occurring variants thereof such asmutant forms associated with gastroschisis or isoforms resulting fromalternative splicing of exons of the ACLP gene.

[0006] The invention includes a nucleic acid molecule which contains thenucleotide sequence of human ACLP cDNA (SEQ ID NO: 1). A nucleic acidmolecule which contains nucleotides 140-3613 (ACLP coding sequence),inclusive, of SEQ ID NO: 1 or a degenerate variant thereof, is alsowithin the invention. Nucleotides 214-3613 encode an ACLP which lacksthe first 25 residues (a putative signal peptide). Preferably, thenucleic acid molecule contains a nucleotide sequence encoding apolypeptide having an amino acid sequence that is at least 87% identicalto the sequence of SEQ ID NO: 2. More preferably, the sequence is atleast 90% identical to SEQ ID NO: 2, more preferably at least 95%, morepreferably at least 98%, more preferably at least 99%, and mostpreferably, the nucleotide sequence encodes a polypeptide the amino acidsequence of which is SEQ ID NO: 2.

[0007] An isolated nucleic acid molecule containing a strand whichhybridizes at high stringency to a DNA having the sequence of SEQ ID NO:1, or the complement thereof is also within the invention. The nucleicacid molecule may be a primer useful to amplify ACLP DNA in a polymerasechain reaction (PCR). For example, the nucleic acid is at least 5nucleotides but less than 50 nucleotides in length. Alternatively, thenucleic acid molecule may encompass the entire coding sequence of ACLPcDNA, i.e., nucleotides 140-3613, inclusive, of SEQ ID NO: 1.Preferably, the nucleic acid molecule spans a gastroschisis-associatedmutation in an ACLP gene. Such a molecule is useful as a hybridizationprobe to identify a genetic alteration, e.g., a deletion, duplication,point mutation, or translocation, that indicates that an individual hasgastroschisis, is predisposed to developing gastroschisis, or is aheterozygous carrier of a genetic alteration associated withgastroschisis.

[0008] By “isolated nucleic acid molecule” is meant a nucleic acidmolecule that is free of the genes which, in the naturally-occurringgenome of the organism, flank an ACLP gene. The term therefore includes,for example, a recombinant DNA which is incorporated into a vector; intoan autonomously replicating plasmid or virus; or into the genomic DNA ofa procaryote or eucaryote; or which exists as a separate molecule (e.g.,a cDNA or a genomic or cDNA fragment produced by PCR or restrictionendonuclease digestion) independent of other sequences. It also includesa recombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence. The term excludes large segments of genomic DNA,e.g., such as those present in cosmid clones, which contain an ACLP geneflanked by one or more other genes which naturally flank it in anaturally-occurring genome.

[0009] Nucleic acid molecules include both RNA and DNA, including cDNA,genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Wheresingle-stranded, the nucleic acid molecule may be a sense strand or anantisense strand. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprocaryote or eucaryote at a site other than its natural site; or whichexists as a separate molecule (e.g., a cDNA or a genomic or cDNAfragment produced by polymerase chain reaction (PCR) or restrictionendonuclease digestion) independent of other sequences. It also includesa recombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

[0010] Hybridization is carried out using standard techniques such asthose described in Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, (1989). “High stringency” refers to DNAhybridization and wash conditions characterized by high temperature andlow salt concentration, e.g., hybridization and wash conditions of 650 Cat a salt concentration of 0.1× SSC. “Low” to “moderate” stringencyrefers to DNA hybridization and wash conditions characterized by lowtemperature and high salt concentration, e.g. wash conditions of lessthan 60° C. at a salt concentration of at least 1.0× SSC. For example,high stringency conditions may include hybridization at 42° C. in asolution containing 50% formamide; a first wash at 65° C. using asolution of 2× SSC and 1% SDS; followed by a second wash at 65° C. usinga solution of 0.1%× SSC. Lower stringency conditions suitable fordetecting DNA sequences having about 50% sequence identity to an ACLPgene are detected by, for example, hybridization at 42° C. in theabsence of formamide; a first wash at 2° C. in a solution of 6× SSC and1% SDS; and a second wash at 50° C. in a solution of 6× SSC and 1% SDS.TABLE 1 Human ACLP cDNA (SEQ ID NO:1) 1 tccctcgctc accccatcct ctctcccgccccttcctgga ttccctcacc cgtctcgatc 61 ccctctccgc cctttcccag agacccagagcccctgaccc cccgcgccct ccccggagcc 121 ccccgcgcgt gccgcggcca tggcggccgtgcgcggggcg cccctgctca gctgcctcct 181 ggcgttgctg gccctgtgcc ctggagggcgcccgcagacg gtgctgaccg acgacgagat 241 cgaggagttc ctcgagggct tcctgtcagagctagaacct gagccccggg aggacgacgt 301 ggaggccccg ccgcctcccg agcccaccccgcgggtccga aaagcccagg cggggggcaa 361 gccagggaag cggccaggga cggccgcagaagtgcctccg gaaaagacca aagacaaagg 421 gaagaaaggc aagaaagaca aaggccccaaggtgcccaag gagtccttgg aggggtcccc 481 caggccgccc aagaagggga aggagaagccacccaaggcc accaagaagc ccaaggagaa 541 gccacctaag gccaccaaga agcccaaggaggagccaccc aaggccacca agaagcccaa 601 agagaagcca cccaaggcca ccaagaagcccccgtcaggg aagaggcccc ccattctggc 661 tccctcagaa accctggagt ggccactgcccccacccccc agccctggcc ccgaggagct 721 accccaggag ggaggggcgc ccctctcaaataactggcag aatccaggag aggagaccca 781 tgtggaggca caggagcacc agcctgagccggaggaggag accgagcaac ccacactgga 841 ctacaatgac cagatcgaga gggaggactatgaggacttt gagtacattc ggcgccagaa 901 gcaacccagg ccacccccaa gcagaaggaggaggcccgag cgggtctggc cagagccccc 961 tgaggagaag gccccggccc cagccccggaggagaggatt gagcctcctg tgaagcctct 1021 gctgcccccg ctgccccctg actatggtgatggttacgtg atccccaact acgatgacat 1081 ggactattac tttgggcctc ctccgccccagaagcccgat gctgagcgcc agacggacga 1141 agagaaggag gagctgaaga aacccaaaaaggaggacagc agccccaagg aggagaccga 1201 caagtgggca gtggagaagg gcaaggaccacaaagagccc cgaaagggcg aggagttgga 1261 ggaggagtgg acgcctacgg agaaagtcaagtgtcccccc attgggatgg agtcacaccg 1321 tattgaggac aaccagatcc gagcctcctccatgctgcgc cacggcctgg gggcacagcg 1381 cggccggctc aacatgcaga ccggtgccactgaggacgac tactatgatg gtgcgtggtg 1441 tgccgaggac gatgccagga cccagtggatagaggtggac accaggagga ctacccggtt 1501 cacaggcgtc atcacccagg gcagagactccagcatccat gacgattttg tgaccacctt 1561 cttcgtgggc ttcagcaatg acagccagacatgggtgatg tacaccaacg gctatgagga 1621 aatgaccttt catgggaacg tggacaaggacacacccgtg ctgagtgagc tcccagagcc 1681 ggtggtggct cgtttcatcc gcatctacccactcacctgg aatggcagcc tgtgcatgcg 1741 cctggaggtg ctggggtgct ctgtggcccctgtctacagc tactacgcac agaatgaggt 1801 ggtggccacc gatgacctgg atttccggcaccacagctac aaggacatgc gccagctcat 1861 gaaggtggtg aacgaggagt gccccaccatcacccgcact tacagcctgg gcaagagctc 1921 acgaggcctc aagatctatg ccatggagatctcagacaac cctggggagc atgaactggg 1981 ggagcccgag ttccgctaca ctgctgggatccatggcaac gaggtgctgg gccgagagct 2041 gttgctgctg ctcatgcagt acctgtgccgagagtaccgc gatgggaacc cacgtgtgcg 2101 cagcctggtg caggacacac gcatccacctggtgccctca ctgaaccctg atggctacga 2161 ggtggcagcg cagatgggct cagagtttgggaactgggcg ctgggactgt ggactgagga 2221 gggctttgac atctttgaag atttcccggatctcaactct gtgctctggg gagctgagga 2281 gaggaaatgg gtcccctacc gggtccccaacaataacttg cccatccctg aacgctacct 2341 ttcgccagat gccacggtat ccacggaggtccgggccatc attgcctgga tggagaagaa 2401 ccccttcgtg ctgggagcaa atctgaacggcggcgagcgg ctagtatcct acccctacga 2461 tatggcccgc acgcctaccc aggagcagctgctggccgca gccatggcag cagcccgggg 2521 ggaggatgag gacgaggtct ccgaggcccaggagactcca gaccacgcca tcttccggtg 2581 gcttgccatc tccttcgcct ccgcacacctcaccttgacc gagccctacc gcggaggctg 2641 ccaagcccag gactacaccg gcggcatgggcatcgtcaac ggggccaagt ggaacccccg 2701 gaccgggact atcaatgact tcagttacctgcataccaac tgcctggagc tctccttcta 2761 cctgggctgt gacaagttcc ctcatgagagtgagctgccc cgcgagtggg agaacaacaa 2821 ggaggcgctg ctcaccttca tggagcaggtgcaccgcggc attaaggggg tggtgacgga 2881 cgagcaaggc atccccattg ccaacgccaccatctctgtg agtggcatta atcacggcgt 2941 gaagacagcc agtggtggtg attactggcgaatcttgaac ccgggtgagt accgcgtgac 3001 agcccacgcg gagggctaca ccccgagcgccaagacctgc aatgttgact atgacatcgg 3061 ggccactcag tgcaacttca tcctggctcgctccaactgg aagcgcatcc gggagatcat 3121 ggccatgaac gggaaccggc ctatcccacacatagaccca tcgcgcccta tgacccccca 3181 acagcgacgc ctgcagcagc gacgcctacaacaccgcctg cggcttcggg cacagatgcg 3241 gctgcggcgc ctcaacgcca ccaccaccctaggcccccac actgtgcctc ccacgctgcc 3301 ccctgcccct gccaccaccc tgagcactaccatagagccc tggggcctca taccgccaac 3361 caccgctggc tgggaggagt cggagactgagacctacaca gaggtggtga cagagtttgg 3421 gaccgaggtg gagcccgagt ttgggaccaaggtggagccc gagtttgaga cccagttgga 3481 gcctgagttc gagacccagc tggaacccgagtttgaggaa gaggaggagg aggagaaaga 3541 ggaggagata gccactggcc aggcattccccttcacaaca gtagagacct acacagtgaa 3601 ctttggggac ttctgagatc agcgtcctaccaagacccca gcccaactca agctacagca 3661 gcagcacttc ccaagcctgc tgaccacagtcacatcaccc atcagcacat ggaaggcccc 3721 tggtatggac actgaaagga agggctggtcctgccccttt gagggggtgc aaacatgact 3782 gggacctaag agccagaggc tgtgtagaggctcctgctcc acctgccagt ctcgtaagag 3841 atggggttgc tgcagtgttg gagtaggggcagagggaggg agccaaggtc actccaataa 3901 aacaagctca tggcaaaaaa aaaaaaaaaaaaaaa

[0011] The invention also includes a substantially pure human ACLPpolypeptide. A substantially pure ACLP polypeptide may be obtained, forexample, by extraction from a natural source (e.g., a vascular smoothmuscle cell); by expression of a recombinant nucleic acid encoding anACLP; or by chemically synthesizing the protein. A polypeptide orprotein is substantially pure when it is separated from thosecontaminants which accompany it in its natural state (proteins and othernaturally-occurring organic molecules). Typically, the polypeptide issubstantially pure when it constitutes at least 60%, by weight, of theprotein in the preparation. Preferably, the protein in the preparationis at least 75%, more preferably at least 90%, and most preferably atleast 99%, by weight, ACLP. A substantially pure ACLP may be obtained,for example, by extraction from a natural source (e.g., a vascularsmooth muscle cell); by expression of a recombinant nucleic acidencoding an ACLP; or by chemically synthesizing the protein. Purity canbe measured by any appropriate method, e.g., column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis. Accordingly,substantially pure polypeptides include recombinant polypeptides derivedfrom a eucaryote but produced in E. coli or another procaryote, or in aeucaryote other than that from which the polypeptide was originallyderived.

[0012] For expression of recombinant ACLP, an ACLP-encoding nucleicacidc is operably linked to a regulatory sequence, e.g., a promoter. By“promoter” is meant a minimal DNA sequence sufficient to directtranscription. Promoters may be constitutive or inducible, and may becoupled to other regulatory sequences or “elements” which renderpromoter-dependent gene expression cell-type specific, tissue-specificor inducible by external signals or agents; such elements may be locatedin the 5′ or 3′ region of the native gene, or within an intron. DNAencoding an ACLP may be operably linked to such regulatory sequences forexpression of the polypeptide in procaryotic or eucaryotic cells. By“operably linked” is meant that a coding sequence and a regulatorysequence(s) are connected in such a way as to permit gene expressionwhen the appropriate molecules (e.g., transcriptional activatorproteins) are bound to the regulatory sequence(s).

[0013] To produce recombinant ACLP, a cell containing an ACLP-encodingsequence operably linked to appropriate regulatory sequences is culturedunder conditions permitting expression of a nucleic acid molecule. Thecell may be a procaryotic cell or a eucaryotic cell. To obtainpost-translationally modified, e.g., glycosylated recombinant ACLP, therecombinant polypeptide is produced in a eucaryotic cell, e.g., a yeastor mammalian cell.

[0014] An ACLP preferably contains an amino acid sequence that is atleast 87% identical to the amino acid sequence of SEQ ID NO: 2. Morepreferably, the amino acid sequence is at least 90% (more preferably atleast 95%, more preferably at least 98%, more preferably at least 99%)identical to SEQ ID NO: 2. Most preferably, the polypeptide contains theamino acid sequence of SEQ ID NO: 2.

[0015] The invention also includes polypeptides which contain a portionof naturally-occurring ACLP, e.g., an ACLP fragment containing alysine-rich/proline rich domain (amino acids 117-164 of SEQ ID NO: 2),an ACLP fragment containing a discoidin-like domain (amino acids 385-540of SEQ ID NO: 2), or an ACLP fragment containing a carboxypeptidase-likedomain (amino acids 62-969 of SEQ ID NO: 2).

[0016] Where a particular polypeptide or nucleic acid molecule is saidto have a specific percent identity to a reference polypeptide ornucleic acid molecule of a defined length, the percent identity isrelative to the reference polypeptide or nucleic acid molecule. Thus, apeptide that is 50% identical to a reference polypeptide that is 100amino acids long can be a 50 amino acid polypeptide that is completelyidentical to a 50 amino acid long portion of the reference polypeptide.It might also be a 100 amino acid long polypeptide which is 50%identical to the reference polypeptide over its entire length. Ofcourse, many other polypeptides will meet the same criteria. The samerule applies for nucleic acid molecules.

[0017] For polypeptides, the length of the reference polypeptidesequence will generally be at least 10 amino acids, preferably at least20 amino acids, more preferably at least 25 amino acids, and mostpreferably 35 amino acids, 50 amino acids, or 100 amino acids. Fornucleic acids, the length of the reference nucleic acid sequence willgenerally be at least 25 nucleotides, preferably at least 50nucleotides, more preferably at least 75 nucleotides, and mostpreferably 100 nucleotides or 300 nucleotides.

[0018] In the case of polypeptide sequences which are less than 100%identical to a reference sequence, the non-identical positions arepreferably, but not necessarily, conservative substitutions for thereference sequence. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine andglutamine; serine and threonine; lysine and arginine; and phenylalanineand tyrosine.

[0019] Sequence identity can be measured using sequence analysissoftware (for example, the Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705), with the defaultparameters as specified therein. TABLE 2 Human ACLP amino acid sequence(SEQ ID NO:2) MAAVRGAPLLSCLLALLALCPGGRPQTVLTDDEIEEFLEGFLSELEPEPREDDVEAPPPPEPTPRVRKAQAGGKPGKRPGTAAEVPPEKTKDKGKKGKKDKGPKVPKESLEGSPRPPKKGKEKPPKATKKPKEKPPKATKKPKEEPPKATKKPKEKPPKATKKPPSGKRPPILAPSETLEWPLPPPPSPGPEELPQEGGAPLSNNWQNPGEETHVEAQEHQPEPEEETEQPTLDYNDQIEREDYEDFEYIRRQKQPRPPPSRRRRPERVWPEPPEEKAPAPAPEERIEPPVKPLLPPLPPDYGDGYVIPNYDDMDYYFGPPPPQKPDAERQTDEEKEELKKPKKEDSSPKEETDKWAVEKGKDHKEPRKGEELEEEWTPTEKVKCPPIGMESHRIEDNQIRASSMLRHGLGAQRGRLNMQTGATEDDYYDGAWCAEDDARTQWIEVDTRRTTRFTGVITQGRDSSIHDDFVTTFFVGFSNDSQTWVMYTNGYEEMTFHGNVDKDTPVLSELPEPVVARFIRIYPLTWNGSLCMRLEVLGCSVAPVYSYYAQNEVVATDDLDFRHHSYKDMRQLMKVVNEECPTITRTYSLGKSSRGLKIYAMEISDNPGEHELGEPEFRYTAGIHGNEVLGRELLLLLMQYLCREYRDGNPRVRSLVQDTRIHLVPSLNPDGYEVAAQMGSEFGNWALGLWTEEGFDIFEDFPDLNSVLWGAEERKWVPYRVPNNNLPIPERYLSPDATVSTEVRAIIAWMEKNPFVLGANLNGGERLVSYPYDMARTPTQEQLLAAAMAAARGEDEDEVSEAQETPDHAIFRWLAISFASAHLTLTEPYRGGCQAQDYTGGMGIVNGAKWNPRTGTINDFSYLHTNCLELSFYLGCDKFPHESELPREWENNKEALLTFMEQVHRGIKGVVTDEQGIPIANATISVSGINHGVKTASGGDYWRILNPGEYRVTAHAEGYTPSAKTCNVDYDIGATQCNFILARSNWKRIREIMAMNGNRPIPHIDPSRPMTPQQRRLQQRRLQHRLRLRAQMRLRRLNATTTLGPHTVPPTLPPAPATTLSTTIEPWGLIPPTTAGWEESETETYTEVVTEFGTEVEPEFGTKVEPEFETQLEPEFETQLEPEFEEEEEEEKEEEIATGQAFPFTTVET YTVNFGDF

[0020] A substantially pure DNA containing an ACLP promoter/enhancersequence (SEQ ID NO: 3) is useful for directing transcription of DNAencoding all or part of ACLP or of DNA encoding a heterologouspolypeptide (e.g., a polypeptide other than ACLP or an ACLP the sequenceof which corresponds to a naturally-occurring ACLP of a species otherthan the species from which the promoter/enhancer sequence is derived).For example, a murine ACLP promoter/enhancer sequence may be operablylinked to DNA encoding human ACLP for therapeutic expression of ACLP inhuman patients. To regulate transcription of the polypeptide-encodingsequence (e.g., developmental stage-specific transcription), thepromoter/enhancer sequence is operably linked to a polypeptide-encodingsequence. The ACLP promoter/enhancer sequence directs transcription of apolypeptide-encoding sequence.

[0021] By “promoter/enhancer sequence” is meant a DNA sequence located5′ to the transcriptional start site of the ACLP gene and which containsone or more cis-acting elements which regulate transcription, e.g., cellspecific transcription. The elements may be contiguous or separated byDNA not involved in the regulation of transcription, e.g., an enhancerelement may be in a position immediately adjacent to the promoterelement or up to several kilobases upstream or downstream of thetranscriptional start site. The promoter/enhancer DNA is preferablyderived from the 5′ region of a mammalian ACLP gene, such as that of themouse (SEQ ID NO: 3), and regulates expression of a polypeptide-encodingDNA to which it is operably linked. The promoter/enhancer sequenceregulates developmental stage-specific expression, e.g., expression inembryonic cells, of a polypeptide-encoding sequence. TABLE 3 Mouse ACLPpromoter/enhancer (SEQ ID NO:3)AAGCTTAGTCTCCCTCTCTCCTGGCTCCTCTCCTGGGGCTTCCCTATGGAGGTAGCACTTACAGAAGATGCTTGTTCCAAACCTTCAGGGGTACAAACTACACAGATATACTGAAGGACAGGAGGCTGGGGCCTCCCCCCACCCCCAACAGCCACTGTTCTCTCAGGAGCTCTGCTTCTGCTCTGCAGCATTGAAAACAAAACTGAAGGACACCTTCCTTCTCTCAGGCCAGCCCAGTGCTGTTGTGTGATCCCTCGGGAAGACTCTAACGCATTCACAGGGACAACAGGAGTTGGGAGGGAGAGGAGTTACAGAACTTTCCAGCAGGACCTCAGGAGAACGCCTGGACACGGACAGGAACCCCCAACCCCTCAGGGACCCCCTTGGACCCTTTGAGTGCTCCTGATCATGGAAGCCACCAGCCTCCCGATTCCTCAGCTGTGGCCTTGGCAGTGCCCTCTGGACATTTGACTTAAACGCTATGCTCTTCAGCAGAGTGGAGAGCTCTCCTCACAGGCTCTGGCTTCTGGTTGTCCTCTTGCCCCAGCGCTGTGGGCCCAGGTTAGAAAGACTTCCTGAGGACAGGCTCCCTCAGGAGGATCCCCAGCGTACGACTGTGCTCCCACGCACCTTTCCGGATTTTCTGTGTGGAGGCCTCAACCCCTCAGGCCTCCTGGGCCAGCTCCTCTGCTCGAATTCCTGTCCGTGACTCATTGAGGCTCAGGAAAAGGCTTTCTAGACCTTAGGTTTCTTTGTTTTCCATTTTTGAAATGGCTTCTGTTTTCCCTGGCAGAGAATATCCAACCCAAATTCAGTCCAAGTATGACCCATGCCTAGGGAAGTGACATCCATGTCCCCTCATGCACCCTGTGGCATACCCAGCATGACACACTGGACCAGACTGGGGGCACGGAAGCCAATTCCCAGAACTGACTTTGAGCACAATGATTCAGAGGGTGACCATGAGTGAGACTTGCTTTACTCTTGCTCTGCGACCAGGTTGAAGTCTCTCATGGGGAGGCCTAGCTGTGAGAGGATTGTCCTGGGATGGGGGAAGGGGGAGCAAAGTGGATGAGGACCAACAGCCTGTGGGATGCAAGGGCTGATCGTGTGTGCTAGGCACAGCACAAAGTGGTCCATTTAGCCGGGCAGTGGTGGTGCACACCTTTAATCCCAGCACTTGGGAGGCATCAGCAGGTGGGTTTCTGAGTTCGAGGCCAGCCTGGTCTACAGAGCAAGTTCCAGGACAGCCAGAGCTACACAGAGAAACTCTGTCTCAAAAAAATCGAATAAACCAGAAAGGTGGTCCATTTAATATGCGTATAGTAAGTTGTGGACACGGGAGTTCCCCTGCTGAGTCAGACAGCTAGGAGGGCTAAGATGGGTTAGACCCTCCCCCCCCCCACACACACACACACACACTCACACACACATCAGTTCTTGGCATAGTCTCCATGCTTCCTCAAGGAGAGCCAGAAAGGAGACTGCCGGGAGGAGCTTGCCTACTCCCTGAGAGCAGTGGGTTACAGAGCCCAGTGCCCGAAAATTTCCCCTTTTTCTCCCTGCTCATGCTGGACAGAGAGGGTGAGGGTGAGGGTGAAAGACTGAGGAGGTGGCATCGTGTTGGTGTTTCTTGACCTGCTTTTTCTTTTTTCTCTTCCAGCTGAGATGTAAACTTTCCCATGTCAATCATCTGGGGGTCGCTATTCTTTTTTATCAGAGTGCCTCCCCACCTTGGTTGAAAGCTGCCTGCCACTACCCTGGACCTATGGCTGCTACAAGCCCACGTTCACATCTTTAATCCTTCATGGGTAAATGCTCTGGCATTCCTGGGCTTAGCTATGATGGCCATTATGAGCCAGCCAACGTTTGTATTCTAGAAGCCATAGCTGAAGCTGTTGTAAACAATTTGTTGTTTTAACCGCTTCTGGTCAGAGGAAGGAGAGAATAGCTATTACTCCACATTGGGACCTGAGCCCTGAGCTCTGAAGTGGGGCTCCTATCTCCATAAGGACAGCAGCTTGCTGAGAACAGCTTTTCACAGCCTTCCTCGCAAAAATTGGCTCCAAAGACCTGGGATGTTGGTGATAACTGGACAAAGGTGACACCTGTGCAAGCACACAGCAGGTGACACTTTGAAGAGCTAACCTCCAGAAAGTGGAAAGGAGGTGATCGCCAGTACCCTCGAGGGCCCTACTCCCTCCCTCCCCTAGCAATCTCCCTGGGCTCAGAGCAAAGGGCACAGCGGGTTAGAGCACAGGTCTCCTTAGACTCCCCACACTCCCTTCCCCATAACTGTTGCATTCTTTTCTCCCAGGCCTTCCTCCCCGCTAGGCGCCCTGCACCCACACCCTCTAAACTGGCGCGTGACGCTGCTATTAGTCTGGGCTCCGTGCTGTCCGCCTCCCTCCCCCGCAGCCCCCGGTCCAAGGCCGGCTCCTCCTCCTCCCCCTCCGGAAACCCGAAGCCCCCGCCCCGGCCAGGCCGTCGCAAGCGCTCTGGAGGGCGGTCCGCGTGAGAGCCAGCCACGCGGGGCAGGAGCGCCCAGTTGCTGCCGGAGCTGGGCCCGCCAGAACCTCTCCTGGAGCCCCTTGCTCTCCTTGAATCTCCCTTTCCCACCGCTTTCTGGATACCCTTGACGCCCACGTTCCTCGCGCCCTTTCCCGCCCCTACGCGGGGCGCTGCCCCTGCCACCCAAGTCCCTGCTCAAGCCCGCCCGGTCCCGCGCGTGCCCAGAGCCATG

[0022] The invention also includes a vector containing thepromoter/enhancer DNA of the invention (operably linked to apolypeptide-encoding DNA sequence), and a vascular smooth muscle cellcontaining the vector. Also within the invention is a method ofdirecting vascular smooth cell-specific expression of the polypeptide byintroducing the vector into a vascular smooth muscle cell andmaintaining the cell under conditions which permit expression of thepolypeptide, e.g., introducing the vector into a human patient for genetherapy.

[0023] A method of detecting a gastroschisis-associated geneticalteration is carried out by providing a sample of DNA or RNA from apatient or fetus, and determining whether the DNA or RNA contains amutation in a gene encoding an ACLP. Detection of such an ACLP mutationindicates that the patient or fetus has a genetic alteration that isassociated with the development of gastroschisis. The presence of agastroschisis-associated genetic alteration is diagnostic ofgastroschisis or a predisposition to developing gastroschisis. Themethod can also be used to identify heterozygous carriers of a mutationassociated with gastroschisis. Such individuals may be asymptomatic butare at risk of having children which are homozygous for an ACLP mutation(and therefore, likely to develop clinical gastroschisis). Tissuesamples from adult patients are obtained by conventional means, e.g.,biopsy or venipuncture. Prenatal testing is carried out by obtainingfetal tissue samples, e.g., by amniocentesis or chorionic villisampling.

[0024] Patient-derived DNA is examined for genetic abnormalities in theACLP gene, e.g., by detecting restriction fragment length polymorphisms(RFLPs), deletions, point mutations, or other defects. The diagnosticmethod includes the step of subjecting the sample to polymerase chainreaction (PCR), using a forward PCR primer complementary to a portion ofthe antisense strand of the gene, the portion being within (a) a firstintron of the gene, or (b) the 5′ untranslated region adjacent to thestart codon of the gene; and a reverse PCR primer complementary to afragment of the sense strand of the gene, this fragment being within (a)a second intron of the gene, or (b) the 3′ untranslated region adjacentto the termination codon of the gene. PCR can also be used to detectmutations in an ACLP promoter or other regulatory sequences usingprimers that flank the mutation. ACLP mutations and/or aberrant ACLPexpression can also be detected using standard hybridization techniques,such as Northern blotting.

[0025] Fragments of ACLP are useful to raise ACLP-specific antibodies.Accordingly, the invention includes an antibody, e.g., a polyclonalantisera or a monoclonal antibody preparation, that selectively binds toan ACLP. ACLP-specific antibodies are used to diagnose gastroschisis ora predisposition thereto. For example, a diagnostic method is carriedout by providing a tissue sample from a patient or fetus, and detectingexpression of an ACLP gene in the tissue sample. Expression is measuredby detecting the amount of ACLP-specific antibody that binds to thetissue sample, e.g., by ELISA assay, Western blot assay, orimmunohistochemical staining of tissue sections. Expression of ACLP isalso measured by detecting the level of ACLP transcript in the tissuesample. Regardless of the method of detection of ACLP expression, areduction in the amount of expression in the patient-derived tissuesample compared to the level of expression in a normal control tissuesample indicates that the patient or fetus from which the sample wasobtained has or is predisposed to developing gastroschisis.

[0026] Methods of treating or preventing the development ofgastroschisis are also within the invention. For example, one treatmentregimen includes the steps of identifying a patient with or at risk ofdeveloping gastroschisis, and introducing into cells of the patient anisolated nucleic acid encoding ACLP, e.g., a nucleic acid which containsthe nucleotide sequence of 140 to 3613 of SEQ ID NO: 1. The cells intowhich the DNA was introduced produce the recombinant ACLP to compensatefor a gastroschisis-associated genetic alteration, e.g., a mutationresulting in reduced production of ACLP or a mutation resulting in theproduction of a defective ACLP. Rather than administering ACLP-encodingDNA to the patient, an ACLP (e.g., a polypeptide having the sequence ofSEQ ID NO: 2) or a fragment thereof may be introduced into the patient.

[0027] An animal model for gastroschisis is useful to study thedevelopment of the condition as well as to evaluate therapeuticapproaches to treatment or prevention of gastroschisis. Agenetically-altered non-human mammal, all diploid cells of which containa mutation in an endogenous gene encoding an ACLP, is included in theinvention. For example, a mammal with a homozygous null mutation in itsACLP gene(s) develops gastroschisis. Preferably, the mammal is a rodentsuch as a mouse. The genetically altered non-human mammal producesaltered levels of ACLP or mutant forms of ACLP. The levels of ACLP geneproduct in the genetically altered mammal can be increased or decreasedat different time periods during development. By “genetically alteredmammal” is meant a mammal in which the genomic DNA sequence has beenmanipulated in some way. The genetically altered mammal may be aknockout in which the endogenous ACLP sequences have been deleted orotherwise altered to decrease or change the pattern of expression.Alternatively, the genetically altered mammal may be transgenic. Forexample, the transgenic mammal may express ACLP sequences from anotherspecies, may overexpress ACLP gene product, or may express ACLP intissues and at developmental stages other than those in which ACLP isexpressed in a wild type animal.

[0028] The nucleated cells of a genetically altered mammal not producinga functional endogenous ACLP may be engineered to encode a human ACLP,and to express functional human ACLP, or, alternatively, ACLP fromanother heterologous species.

[0029] Preferably, the genetically altered non-human mammal is a rodentsuch as a mouse or a rat, the germ cells and somatic cells of whichcontain a mutation in DNA encoding ACLP. All diploid cells of such ananimal contain a mutation in one or both alleles of the endogenous ACLPgene. The mutation can, for example, be a deletion, an insertion, or anucleotide substitution. The mutation could be in the ACLP regulatoryregions or in the coding sequence. It can, e.g., introduce a stop codonthat results in production of a truncated, inactive gene product or itcan be a deletion of all or a substantial portion of the codingsequence. For example, one or more exons, e.g., exons 7-15, of an ACLPgene may be deleted. By the term “null mutation” is meant a mutationthat reduces the expression or activity level of the protein encoded bythe mutated gene by more than 80% relative to the unmutated gene. Amouse harboring such a null mutation is a knockout mouse. An ACLPknockout mouse, i.e., one that harbors a homozygous ACLP null mutation,has been found to have an abdominal defect with an extrusion ofabdominal organs i.e., gastroschisis.

[0030] The invention also includes a mammalian cell line, e.g.,immortalized ACLP deficient cells, the genomic DNA of which contains anull mutation in DNA encoding ACLP. Such cells lack the ability tosynthesize full length functional ACLP. The cells harboring the nullmutation may be derived from a cell obtained from a ACLP deficientmammal, e.g., an ACLP knockout mouse.

[0031] Compounds capable of promoting expression or function of an ACLPmay be therapeutically useful to treat gastroschisis. Accordingly, theinvention includes a method of screening a candidate compound toidentify a compound capable of stimulating expression of an ACLP, e.g.,human ACLP, by (a) providing a cell or tissue expressing capable ofexpressing a ACLP, (b) contacting the cell or tissue with the candidatecompound, and (c) determining the amount of expression of the ACLP bythe cell. An increase in the amount of ACLP expression in the presenceof the candidate compound compared to that in the absence of thecandidate compound indicates that the compound stimulates expression ofthe ACLP.

[0032] In addition to diagnostic methods, such as described above, thepresent invention encompasses methods and compositions for evaluatingappropriate treatment, and treatment effectiveness of pathologicalconditions associated with aberrant expression of ACLP. For example, theACLP gene can be used as a probe to classify cells in terms of theirlevel of ACLP expression, or as a source of primers for diagnostic PCRanalysis in which mutations and allelic variation of ACLP can bedetected.

[0033] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWING

[0034]FIG. 1A is a diagram showing a comparison of deduced open readingframes of human ACLP and mouse ACLP. The human and mouse proteinscontain 1158 and 1128 amino acids, respectively. Highlighted motifsinclude a signal peptide (bold, underline), a 4-fold lysine- andproline-rich repeating motif (bold, italic), a discoidin-like domain(bold, italic, underline), and a region with homology to thecarboxypeptidases (bold).

[0035]FIG. 1B is a diagram showing the location of peptide domains ofhuman ACLP. The signal peptide sequence at the N-terminus is designated“Signal”; the 4-fold repeating motif is designated “Repeat”; thediscoidin-like domain is designated “DLD”; and the region with homologyto carboxypeptidases is designated “CLD”.

[0036]FIG. 2A is a diagram of mouse genomic DNA showing a map of themouse ACLP gene and neighboring DNA polymerase delta small subunit gene.

[0037]FIG. 2B is a diagram of a restriction map of genomic DNAcontaining the mouse ACLP gene and neighboring DNA polymerase deltasmall subunit gene.

[0038]FIG. 3 is a diagram showing a map of the targeting construct usedto make an ACLP knockout mouse.

DETAILED DESCRIPTION

[0039] A mutation in an ACLP-encoding nucleic acid resulting in adecrease in production of an ACLP compared to the level of ACLPproduction in an animal lacking the mutation has now been shown toresult in the development of gastroschisis in newborn mice. Thefollowing examples describe the cloning and characterization of humanACLP and methods of diagnosing and treating gastroschisis the underlyingdefect of which is a genetic alteration in the ACLP genes.

EXAMPLE 1 Cloning of ACLP Genes

[0040] ACLP was identified in a screen for proteins interacting with theE47 product of the E2A gene. A recombinant E47 fusion protein(N3-SH[ALA]), containing the basic helix loop helix domain of hamstershPan-1 (amino acids 509-646, with mutations R551A, V552L, and R553A)with a heart muscle kinase recognition sequence and the FLAG epitope,was expressed and purified as described (Blanar et al., 1995, Proc.Natl. Acad. Sci. USA 92:5870-4; Blanar and Rutter, 1992, Science256:1014-8). N3-SH[ALA] was phosphorylated by heart muscle kinase in thepresence of γ-³²P-ATP and used to screen a human aorta λgt11 cDNAexpression library (Clonetech) by interaction cloning (Blanar et al.,1995, Proc. Natl. Acad. Sci. USA 92:5870 4; Blanar and Rutter, 1992,Science 256:1014-8). A 1450-bp cDNA clone (ΔE2A-BP) obtained frominteraction cloning was radiolabeled by random priming and used toisolate a 2786 bp cDNA clone from the same human aorta λgt11 cDNAlibrary. Data from Northern blotting experiments revealed that theACLP-1 RNA was about 3.9 kb in size and suggested that the 2786 bp cDNAclone was a partial cDNA clone. Additional 5′ sequences of the ACLP cDNAwere isolated by 5′ rapid amplification of cDNA ends from human aorticsmooth muscle cell RNA (Gibco-BRL). The full length sequence of thehuman ACLP cDNA was found to be 3935 bp and is shown in Table 1 (SEQ IDNO: 1). The full length human ACLP cDNA contains an open reading frame(nucleotides 140-3613 of SEQ ID NO: 1) encoding a polypeptide of 1158amino acids. The open reading frame is preceded by a Kozak consensustranslation initiation sequence, which in turn is preceded by an inframe stop codon.

[0041] The human ACLP protein has a calculated molecular mass of 130kDa, an estimated pI of 4.8, and contains a putative signal peptidesequence. In addition, it contains an 11 amino acid lysine- andproline-rich motif repeated four times at the N-terminus, a domain with30% amino acid identity to the slime mold adhesion protein discoidin I,and a C-terminal domain with 39% identity to carboxypeptidase E. Thehuman ACLP gene maps to the short arm of chromosome 7 (between D7S478amd D7S519).

[0042] The sequence of the human ACLP cDNA (GENBANK™ accession numberAF053944) was compared to sequences present in GENBANK™ databases. A 3′portion of ACLP cDNA was found to share homology with the sequence of acDNA encoding mouse adipocyte enhancer binding protein 1 (AEBP1; He etal., 1995, Nature 378:92). AEBP1 was originally identified as a 2.5 kbcDNA that hybridized to a 4 kb band on Northern blot analysis, and waspredicted to encode a 719 amino acid, 79 kDa protein.

[0043] To isolate mouse ACLP cDNA (GENBANK™ accession number AF053943),first strand cDNA from C2C12 mouse myoblast total RNA was synthesized byreverse transcription with the primer 5′ ATCTGGTTGTCCTCAAT 3′ (SEQ IDNO: 4). The nested primer 5′ TGACTCCATCCCAATAG 3′ (SEQ ID NO: 5) and theanchor primer included in the kit for 5′ rapid amplification of cDNAends was then amplified to produce a product of approximately 1400 bp insize. This product was sequenced using standard methods.

[0044] The entire open reading frame of mouse ACLP was then amplifiedfrom C2C12 RNA by reverse transcription PCR (EXPANDLONG™ Template PCRSystem, Boehringer Mannheim, Indianapolis, Ind.). The human and mouseclones were sequenced by the dideoxy nucleotide chain termination methodusing a combination of Sequenase Version 2.0 (Amersham, ArlingtonHeights, Ill.), the Thermo Sequenase ³³P terminator cycle sequencing kit(Amersham), and the Thermo Sequenase fluorescent-labeled cyclesequencing kit with 7-deaza-GTP (Amersham) on a Licor (Lincoln, Nebr.)apparatus.

[0045] Sequencing of the 3633 bp mouse ACLP cDNA fragment, revealed thatit encoded an open reading frame (1128 amino acids) similar to that ofthe full-length human ACLP cDNA, indicating that it is the mouse ACLPhomologue. A comparison of the human and mouse ACLP amino acid sequencesis shown in FIG. 1A. Overall, the two proteins are 85% identical and 90%similar.

EXAMPLE 2 Identification of a Promoter-Enhancer Sequence Associated withthe ACLP Gene

[0046] To identify genomic sequences that mediate tissue specific anddevelopmental expression pattern of the ACLP gene, a region of genomicDNA adjoining the 5′ end of the mouse ACLP coding sequences was isolated(FIGS. 2A and 2B). Portions of this genomic DNA were then used inreporter transfection assays to determine their ability to directexpression of a reporter gene in transfection assays. ACLPpromoter/enhancer DNA was cloned into the pGL2 Basic vector (Stratagene)and transfected into rat aortic smooth muscle cells (RASMC) to measurepromoter activity. Using this assay, a region containing an ACLPpromoter/enhancer sequence was identified and is shown in Table 3 (SEQID NO: 3). ACLP promoter/enhancer DNA was found to have transcriptionalactivity both in vitro (using cultured cells) and in vivo (in atransgenic mouse).

EXAMPLE 3 Generation and Characterization of Antibodies to ACLP Peptides

[0047] A carboxy terminal fragment of mouse ACLP was expressed inbacteria, purified, and used as an immunogen to raise antibodies inrabbits.

[0048] To produce a polyclonal anti-ACLP antibody, a BamHI-EcoRIfragment of mouse ACLP (encoding amino acids 615-1128) was subclonedinto the pRSET C bacterial expression vector (Invitrogen), and theresulting plasmid was transformed into BL21(DE3)pLysS-competent bacteria(Stratagene). Protein expression was induced with 1 mM isopropylβ-D-thiogalactopyranoside for 3 h. Bacteria were sonicated in lysisbuffer (50 mM NaH₂PO₄, 10 mM Tris, pH 8, 100 mM NaCl) containing theprotease inhibitors aprotinin, leupeptin, and phenylmethylsulfonylfluoride. Lysates were clarified by centrifugation at 10,000 g for 15min, and the pellet was resuspended in lysis buffer supplemented with 8M urea. His-tagged proteins were purified with Talon resin (Clontech)and eluted in lysis buffer containing 8 M urea and 100 mM ethylenediamine tetraacetic acid. Proteins were dialyzed against water andmeasured with the Bio-Rad (Hercules, Calif.) protein assay reagent. 100μg of the purified protein was used to immunize New Zealand whiterabbits. Antiserum was collected, titered against the recombinantprotein, and used for immunoblot analysis. Specificity of the antiserumwas determined by using preimmune serum and by competition with arecombinant protein. The same methods are used to raise antibodies tohuman ACLP. The rabbit antisera raised against a portion of mouse ACLPwas found to crossreact with human ACLP.

[0049] Protein extracts from cultured cells were prepared for Westernblotting in extraction buffer (25 mM Tris, pH 7.4, 50 mM NaCl, 0.5%sodium deoxycholate, 2% Nonidet P-40, and 0.2% sodium dodecyl sulfate)containing the protease inhibitors aprotinin, leupeptin, andphenylmethylsulfonyl fluoride. To obtain proteins from mouse tissues,individual organs were homogenized in 25 mM Tris, pH 7.5, 50 mM NaCl,and 10 mM ethylene diamine tetraacetic acid containing proteaseinhibitors (Complete, Boehringer Mannheim). Proteins were measured withthe BCA protein assay kit (Pierce, Rockford, Ill.). After 50 μg aliquotshad been resolved on 6% sodium dodecyl sulfate-polyacrylamide gels (18),proteins were transferred electrophoretically to nitrocellulosemembranes (Schleicher and Schuell, Keene, N.H.) in 48 mM Tris, pH 8.3,39 mM glycine, 0.037% sodium dodecyl sulfate, and 20% methanol transferbuffer. Blots were equilibrated with 25 mM Tris, pH 8, 125 mM NaCl, and0.1% Tween 20 and blocked in the same solution containing 4% nonfat drymilk. Blots were incubated with anti-ACLP serum diluted 1:1000 and thenhorseradish peroxidase-conjugated goat anti-rabbit serum diluted 1:4000.Membranes were processed with an enhanced chemiluminescence reagent (ECLreagent, NEN, Boston, Mass.) and exposed to film.

[0050] By Western blot analysis, this antibody detected a single bandcorresponding to a protein with an apparent mobility of approximately175 kDa in mouse aortic smooth muscle cells (MASMC) extracts. Thisprotein showed a similar migration to a protein generated bytranscription and translation in vitro of a mouse ACLP cDNA clone,providing additional evidence that the isolated human and mouse cDNAclones encode full-length ACLP.

[0051] Monoclonal antibodies can be obtained using full-length human ormouse ACLP or fragments thereof using standard methods, e.g., theprocess described by Milstein and Kohler, 1975, Nature 256:495-97, or asmodified by Gerhard, 1980, Monoclonal Antibodies, Plenum Press, pages370-371. Hybridomas are screened to identify those producing antibodiesthat are specific for an ACLP. Preferably, the antibody will have anaffinity of at least about 10⁸ liters/mole and more preferably, anaffinity of at least about 10⁹ liters/mole.

EXAMPLE 4 Subcellular Localization and Tissue Localization of ACLPProteins

[0052] To assess the subcellular localization of ACLP, a mouse ACLPexpression construct was generated with a c-myc epitope at theC-terminus. The myc epitope was placed at the C-terminus to avoidinterference with signal peptide-mediated processes, e.g., ACLPsecretion mechanisms. To construct a c-myc-tagged ACLP expressionplasmid (pcDNA3.1/ACLP-Myc-His), the open reading frame of mouse ACLPwas amplified with the Expand Long Template PCR System (BoehringerMannheim). A 5′ primer containing an EcoRI site (5′CGGAATTCAGTCCCTGCTCAAGCCCG 3′; SEQ ID NO: 6) and a 3′ primer containinga HindIII site (5′ CGAAGCTTGAAGTCCCCAAAGTTCACTG 3′; SEQ ID NO: 7) wasused, which resulted in the deletion of the endogenous termination codonin the PCR product. The PCR product was then digested with EcoRI andHindIII restriction enzymes and ligated into the EcoRI and HindIII sitesof pcDNA3.1(−)/Myc-His A (Invitrogen). Cells were transfectedtransiently with pcDNA3.1/ACLP-Myc-His by the DEAE-dextran method withminor modifications (Tan et al., Kidney International 46:690, 1994).Twenty-four hours after transfection, cells were trypsinized and platedonto chamber slides (Nunc, Naperville, Ill.) and grown for an additional24 h. Cells were fixed with 4% paraformaldehyde in phosphate-bufferedsaline and immunostained using standard methods. Amonoclonal anti-c-mycprimary antibody (9E10 Ab-1, Oncogene Research Products, Cambridge,Mass.) and a rhodamine-conjugated goat anti-mouse IgG secondary antibodywere used to immunostain the cells. Nuclei were counterstained withHoechst 33258 (1 μg/ml) and visualized with a fluorescence microscope.

[0053] RASMC and A7r5 cells both exhibited strong membrane-associated orcytoplasmic staining. Staining was most intense in the perinuclearregion and was not observed in the nucleus. Various other tissues wereexamined for the presence of ACLP mRNA and protein. Gene expressionstudies confirmed expression in aortic smooth muscle cells, and levelsof ACLP mRNA were found to be high in the whole aorta (includingadventitia) compared to most other tissue types tested, e.g., heart,brain, stomach, thymus, and liver. ACLP message was also detectable incolon and kidney tissue.

[0054] To examine expression of ACLP, extracts from mouse tissues weresubjected to Western blot analysis using anti-ACLP sera. ACLP wasstrongly expressed in the mouse aorta (without adventitia) but not inthe adventitia, heart, liver, skeletal muscle, or kidney. The presenceof ACLP mRNA in the kidney (but absence of protein) indicates that thelevel of ACLP in the cells is regulated at the level of translation ofACLP mRNA into polypeptide.

[0055] To identify cell types expressing ACLP in an adult animal, insitu hybridization was performed on adult rat aorta and skeletal muscleusing known methods. Adult male Sprague-Dawley rats were perfused with4% paraformaldehyde and their organs were removed and sectioned. ACLPmRNA was detected with a [³⁵S]UTP-labeled antisense riboprobesynthesized with SP6 RNA polymerase from a linearized 0.7 kb fragment ofACLP cDNA. As a control, a sense RNA probe was synthesized with T7 RNApolymerase from a linearized ACLP cDNA fragment. The antisense riboprobedetected specific ACLP expression in the smooth muscle cells of theaorta, whereas the control (sense) probe did not. Neither the sense northe antisense probe hybridized to skeletal muscle cells.

EXAMPLE 5 ACLP Expression in Smooth Muscle Cell Differentiation

[0056] ACLP protein expression was examined during vascular smoothmuscle cell growth and differentiation. RASMC and MASMC were isolatedfrom the thoracic aortas of adult male Sprague-Dawley rats and C57Bl/6mice using standard methods. Human aortic smooth muscle cells (HASMC)were purchased from Clonetics (San Diego, Calif.), and rat A7r5 smoothmuscle cells and C2C12 mouse myoblasts were purchased from the AmericanType Culture Collection (Rockville, Md.). Mouse neural crest cells(Monc-1 cells) were cultured on fibronectin-coated plates. RASMC, MASMC,and A7r5 cells were cultured in Dulbecco's modified Eagle's medium with3.7 g/liter glucose (Gibco-BRL, Gaithersburg, Md.) supplemented with 10%fetal bovine serum (Hyclone, Logan, Utah), 4 mML-glutamine, 100 μg/mlstreptomycin, 100 units/ml penicillin, and 10 mM HEPES (pH 7.4). C2C12cells were grown in Dulbecco's modified Eagle's medium supplemented with15% fetal bovine serum, 4 mM L-glutamine, 100 Ag/ml streptomycin, and100 units/ml penicillin. HASMC were cultured in M199 medium (Gibco)supplemented with 20% fetal bovine serum, 4 mM L-glutamine, 100 μg/mlstreptomycin, and 100 units/ml penicillin. Cells were grown at 37° C. ina humidified incubator containing 5% CO₂. MASMC were cultured for 3 daysin 0.4% calf serum containing medium that induces quiescence. RNA andprotein extracts were then prepared from the cells and analyzed.

[0057] The amount of ACLP mRNA was higher (about 2-fold) inserum-starved (quiescent) MASMC than in control cells (normalproliferating MASMC). In RASMC, ACLP mRNA was approximately 3-fold moreabundant in quiescent cells than in their actively proliferatingcounterparts. ACLP protein was also elevated in quiescent MASMC.

[0058] ACLP expression was examined in an in vitro system fordifferentiating smooth muscle cells from a Monc-1 cell line, a mouseline derived from the neural crest. Monc-1 cells differentiate intosmooth muscle cells when tissue culture medium supplemented with chickembryo extract is replaced with differentiation medium. To examine ACLPexpression during the transition of undifferentiated Monc-1 cells tosmooth muscle, the time course of ACLP expression was measured. ACLPmRNA was nearly undetectable in undifferentiated Monc-1 cells. As thecells differentiated, however, ACLP expression increased until it becamemarked at days 4 and 6 after the start of differentiation. Under theseconditions, induction of ACLP appeared to lag behind that of smoothmuscle a-actin, a marker for smooth muscle cells. To compare the levelof ACLP protein in cells treated similarly, protein extracts wereprepared from undifferentiated Monc-1 cells and from cells allowed todifferentiate for 6 days. ACLP protein was not detectable inundifferentiated Monc-1 cells but was expressed highly (day 6) underconditions that promote Monc-1 cell differentiation into smooth musclecells. The abundance of ACLP protein in these cells was similar to thatin MASMC.

[0059] As is described below, the Monc-1 cells (and other cellsexpressing ACLP) can be used to screen for compounds that stimulate atherapeutic increase in ACLP production (e.g., during celldifferentiation and/or fetal development).

EXAMPLE 6 Genetically-Altered Animals

[0060] An ACLP deficient animal, e.g., an ACLP knockout mouse, isproduced as follows.

[0061] The targeting construct was made by deleting exons, e.g., 7-15 ofthe mouse ACLP gene (see FIG. 3). A SalI-BamHI fragment of the ACLP genewas replaced with pPGK-neo to generate the targeting construct.

[0062] The linearized targeting construct (shown in FIG. 3) wastransfected into murine D3 embryonic stem (ES) cells, and a clone withthe correct homologous recombination (yielding the appropriatelydisrupted ACLP gene shown in FIG. 3) injected into blastocysts and usedto generate ACLP chimeric mice using standard methods. The chimeric micewere bred with wild type mice to generate ACLP-mutated heterozyous mice.ACLP-mutated heterozygous mice were born normal. To generate an ACLPknockout mouse (i.e., homozygous for the ACLP mutation), theheterozygous mice were mated. The genotype of newborn mice was evaluatedat 3 weeks. Out of 205 live pups, 74 were found to have the wild typeACLP gene, 113 were found to be heterozygotes, and 18 were found to behomozygous for the ACLP null mutation, i.e., ACLP knockout mice. Thesedata indicate that many of the knockout mice died before or immediatelyafter birth.

[0063] The phenotype of ACLP knockout mice was evaluated duringdevelopment. Cesarean sections were performed to obtain embryos at 18.5embryonic days (El8.5). ACLP-knockout mice were found to have anabdominal defect with extrusion of abdominal organs, whereas the wildtype mice were normal.

[0064] The progress of the development of gastroschisis is evaluated bymating heterozygous ACLP-mutant mice and obtaining embryos at varioustime points, e.g., E18.5, E16.5, E12.5, E10.5, and E8.5. Embryos areexamined at both gross and microscopic levels. Histological evaluationof embryonic tissue, e.g., to follow the formation of theomphalomesenteric arteries, is used to determine the incidence and timeof development of gastroschisis.

[0065] ACLP-deficient animals can be used to screen for compounds totreat or prevent the development of gastroschisis. To determine whethera given compound prevents or reduces the development of gastroschisis indeveloping embryos, the compound is administered to the pregnant animal(e.g., systemically, in utero, or directly to an embryo itself) and theembryos examined as described above. For example, a nucleic acidencoding a full length wild type ACLP gene (or an ACLP gene which maydiffer from the wild type sequence but still retains ACLP function) canbe tested to evaluate the effect of such gene therapy on the developmentof gastroschisis. A reduction in the severity of gastroschisis intreated embryos compared to untreated embryos indicates that thecompound or gene therapy approach to treatment of gastroschisis isclinically beneficial.

[0066] ACLP deficient mice and ACLP deficient cell lines derived fromsuch mice are useful in determining the etiology of gastroschisis andscreening for therapeutic compositions.

EXAMPLE 7 Diagnosis of Disorders Associated with Altered Levels of ACLPExpression or Activity

[0067] The data described herein indicates that an ACLP mutation (e.g.,in ACLP coding or regulatory sequences) is involved in the developmentof gastroschisis. Thus, individuals (e.g., those with a family historyof the disease) can be tested for the presence of a mutated ACLP genewhich may contribute to the development of gastroschisis in children ofan individual harboring a mutated gene. Detection of such a mutationwill permit appropriate genetic counseling of those individualsregarding the risks associated with pregnancy. In addition, such testingcan be used to identify individuals with subclinical gastroschisis orother related gastrointestinal abnormalities. Prenatal testing may becarried out to determine whether a developing fetus is at risk ofdeveloping gastroschisis. Although gastroschisis may be detected atapproximately the second trimester of pregnancy by conventional prenatalultrasound testing, early detection of a genetic abnormality permitsearly intervention, including genetic therapy, which may prevent thedevelopment of the condition or reduce its severity.

[0068] Analysis can be carried out on any suitable genomic DNA sample(e.g., maternal tissue and/or fetal tissue) to be tested. Typically, ablood sample or a sample of placental or umbilical cord cells is tested.A sample of fetal cells can be obtained by amniocentesis or chorionicvilli sampling.

[0069] Standard genetic diagnostic methods are used to detect a mutationin the ACLP gene. For example, PCR (polymerase chain reaction) is usedto identify the presence of a deletion, addition, or substitution of oneor more nucleotides within any one of the exons of ACLP. Following thePCR reaction, the PCR product can be analyzed by methods as describedabove, such as the heteroduplex detection technique based upon that ofWhite et al., 1992, Genomics 12:301-306, or by techniques such ascleavage of RNA-DNA hybrids using RNase A (Myers et al., 1985, Science230:1242-1246); single-stranded conformation polymorphism (SSCP)analysis (Orita et al., 1989, Genomics 10:298-299); and denaturinggradient gel electrophoresis (DGGE; Myers et al., 1987, Methods Enzymol.155:501-527). PCR may be carried out using a primer which adds aG+C-rich sequence (termed a “GC-clamp”) to one end of the PCR product,thus improving the sensitivity of the subsequent DGGE procedure(Sheffield et al., 1989, Proc. Natl. Acad. Sci. USA 86:232-236). If theparticular mutation present in the patient's family is known to haveremoved or added a restriction site, or to have significantly increasedor decreased the length of a particular restriction fragment, a protocolbased upon restriction fragment length polymorphism (RFLP) analysis(perhaps combined with PCR) can be used to identify the genetic defect.

[0070] In addition to evaluating genomic DNA of a patient, an ACLPdefect can be detected by evaluating an ACLP gene product. Unlikegenomic DNA-based diagnostic methods, this approach permits detection ofdefects resulting in a decrease in the level of expression of an ACLPgene (i.e., a defect which does not involve mutations in the codingsequence itself). In addition to detection of a gene product, geneexpression is also measured using mRNA-based methods, such as Northernblots and in situ hybridization (using a nucleic acid probe derived fromthe relevant cDNA), and quantitative PCR.

[0071] An ACLP gene product can be tested for abnormalities, e.g.,differences in the level of expression compared to wild type ACLP,truncation of an ACLP gene product, or deletion of a portion of an ACLPgene product. Deletion ACLP mutants, e.g., those characterized by theloss of an ACLP epitope, can be detected using an ACLP-specificantibody. Western blotting and Northern blotting techniques are used toquantitate the amount of expression of a ACLP in the tissue of interest.For example, an individual who is heterozygous for a genetic defectaffecting level of expression of ACLP may be diagnosed by detectingreduction in the level of expression of this gene in such ahybridization or antibody-based assay, and an individual who ishomozygous may be identified by detection of a comparatively lower levelof expression.

[0072] The diagnostic method of the invention is carried out bymeasuring ACLP gene expression in a tissue, e.g, a biopsy, or in abodily fluid, e.g., blood or plasma. Detection of expression anddetermination of the level of gene expression is measured using methodsknown in the art, e.g., in situ hybridization, Northern blot analysis,or Western blot analysis using ACLP-specific monoclonal or polyclonalantibodies. An decrease in the level of ACLP expression per cell in thetest sample of tissue compared to the level per cell in control tissueindicates that the patient has gastroschisis, is predisposed todeveloping gastroschisis, or is a carrier of a genetic defect associatedwith gastroschisis.

[0073] The diagnostic procedures described above are useful to identifypatients in need of therapeutic intervention to reduce the severity ofor prevent the development of gastroschisis.

EXAMPLE 8 Treatment of Disorders Associated with Altered Levels of ACLPExpression or Activity

[0074] Gene therapy may be carried out by administering to a patient anucleic acid encoding a therapeutic polypeptide, e.g., an ACLP orfragment thereof, by standard vectors and/or gene delivery systems.Suitable gene delivery systems may include liposomes, receptor-mediateddelivery systems, naked DNA, and viral vectors such as herpes viruses,retroviruses, adenoviruses and adeno-associated viruses, among others.

[0075] In addition to a gene delivery system as described above, thetherapeutic composition may include a pharmaceutically acceptablecarrier, e.g., a biologically compatible vehicle such as physiologicalsaline, suitable for administration to an animal. A therapeuticallyeffective amount of a compound is an amount which is capable ofproducing a medically desirable result in a treated animal, e.g., areduction in the severity of gastroschisis or the prevention of thedevelopment of gastroschisis (e.g., in a fetus).

[0076] Parenteral administration, such as intravenous, subcutaneous,intramuscular, and intraperitoneal delivery routes, may be used todeliver the compound. Dosage for any one patient depends upon manyfactors, including the patient's size, body surface area, age, theparticular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. Dosage of the compound to be administered will vary. Apreferred dosage for intravenous administration of nucleic acids is fromapproximately 10⁶ to 10²² copies of the nucleic acid molecule.Compounds, including therapeutic nucleic acids, may be administeredlocally through the uterine wall to the developing fetus using knownmethods.

[0077] ACLPs may be similarly administered, e.g., locally orsystemically, e.g., intravenously, in a pharmaceutically acceptablecarrier such as physiological saline. Standard methods for intracellulardelivery of peptides can be used, e.g. packaged in liposomes. Suchmethods are well known to those of ordinary skill in the art. It isexpected that an intravenous dosage of approximately 1 to 100 μmoles ofthe polypeptide of the invention would be administered per kg of bodyweight per day. The compositions of the invention are useful forparenteral administration, such as intravenous, subcutaneous,intramuscular, and intraperitoneal.

[0078] ACLP encoding DNA is be introduced into target cells of thepatient by standard vectors, e.g., a vector which contains DNA encodingan ACLP operably linked to an ACLP promoter/enhancer sequence. Suitablegene delivery systems may include liposomes, receptor-mediated deliverysystems, naked DNA, and viral vectors such as herpes viruses,retroviruses, and adenoviruses, among others. ACLP DNA under the controlof a strong constitutive promoter may be administered locally using anadenovirus delivery system.

[0079] Drugs which stimulate an-endogenous ACLP promoter may also beadministered as described above to increase the level of expression ACLPin patients in which the underlying clinical defect is a pathologicallylow level of ACLP production.

EXAMPLE 9 Identification of Compounds that Alter ACLP Expression orActivity

[0080] ACLP knockout mice have the clinical manifestations ofgastroschisis. Compositions that ameliorate the symptoms ofgastroschisis or prevent the development of gastroschisis in adeveloping fetus can be identified using ACLP knockout mice. A testcompound is administered to an ACLP knockout mouse. As a control, thecompound is administered to a normal wild type mouse (preferably withthe same genetic background as the ACLP knockout mouse). A reduction inthe severity of gastroschisis in ACLP knockout mice treated with thetest compound compared to control ACLP mice which have not been exposedto the test compound is an indication that the test compound is capableof ameliorating the symptoms of or preventing the development ofgastroschisis.

[0081] Compounds can also be screened by contacting cells in vitro,e.g., VASMC, MASMC, RASMC, Monc-1 cells, or cells derived from an ACLPknockout mouse or from an animal or patient with gastroschisis, with acandidate compound and measuring the level of ACLP expression (oractivity) in the cells. An increase in cellular ACLP expression(compared to the level of expression in the absence of a test compound)indicates that the compound is clinically useful to prevent or treatgastroschisis in which the underlying defect is pathological reductionin the level of ACLP production.

[0082] Other embodiments are within the following claims.

1 8 1 3935 DNA Homo sapiens CDS (140)...(3613) 1 tccctcgctc accccatcctctctcccgcc ccttcctgga ttccctcacc cgtctcgatc 60 ccctctccgc cctttcccagagacccagag cccctgaccc cccgcgccct ccccggagcc 120 ccccgcgcgt gccgcggcc atggcg gcc gtg cgc ggg gcg ccc ctg ctc agc 172 Met Ala Ala Val Arg Gly AlaPro Leu Leu Ser 1 5 10 tgc ctc ctg gcg ttg ctg gcc ctg tgc cct gga gggcgc ccg cag acg 220 Cys Leu Leu Ala Leu Leu Ala Leu Cys Pro Gly Gly ArgPro Gln Thr 15 20 25 gtg ctg acc gac gac gag atc gag gag ttc ctc gag ggcttc ctg tca 268 Val Leu Thr Asp Asp Glu Ile Glu Glu Phe Leu Glu Gly PheLeu Ser 30 35 40 gag cta gaa cct gag ccc cgg gag gac gac gtg gag gcc ccgccg cct 316 Glu Leu Glu Pro Glu Pro Arg Glu Asp Asp Val Glu Ala Pro ProPro 45 50 55 ccc gag ccc acc ccg cgg gtc cga aaa gcc cag gcg ggg ggc aagcca 364 Pro Glu Pro Thr Pro Arg Val Arg Lys Ala Gln Ala Gly Gly Lys Pro60 65 70 75 ggg aag cgg cca ggg acg gcc gca gaa gtg cct ccg gaa aag accaaa 412 Gly Lys Arg Pro Gly Thr Ala Ala Glu Val Pro Pro Glu Lys Thr Lys80 85 90 gac aaa ggg aag aaa ggc aag aaa gac aaa ggc ccc aag gtg ccc aag460 Asp Lys Gly Lys Lys Gly Lys Lys Asp Lys Gly Pro Lys Val Pro Lys 95100 105 gag tcc ttg gag ggg tcc ccc agg ccg ccc aag aag ggg aag gag aag508 Glu Ser Leu Glu Gly Ser Pro Arg Pro Pro Lys Lys Gly Lys Glu Lys 110115 120 cca ccc aag gcc acc aag aag ccc aag gag aag cca cct aag gcc acc556 Pro Pro Lys Ala Thr Lys Lys Pro Lys Glu Lys Pro Pro Lys Ala Thr 125130 135 aag aag ccc aag gag gag cca ccc aag gcc acc aag aag ccc aaa gag604 Lys Lys Pro Lys Glu Glu Pro Pro Lys Ala Thr Lys Lys Pro Lys Glu 140145 150 155 aag cca ccc aag gcc acc aag aag ccc ccg tca ggg aag agg cccccc 652 Lys Pro Pro Lys Ala Thr Lys Lys Pro Pro Ser Gly Lys Arg Pro Pro160 165 170 att ctg gct ccc tca gaa acc ctg gag tgg cca ctg ccc cca cccccc 700 Ile Leu Ala Pro Ser Glu Thr Leu Glu Trp Pro Leu Pro Pro Pro Pro175 180 185 agc cct ggc ccc gag gag cta ccc cag gag gga ggg gcg ccc ctctca 748 Ser Pro Gly Pro Glu Glu Leu Pro Gln Glu Gly Gly Ala Pro Leu Ser190 195 200 aat aac tgg cag aat cca gga gag gag acc cat gtg gag gca caggag 796 Asn Asn Trp Gln Asn Pro Gly Glu Glu Thr His Val Glu Ala Gln Glu205 210 215 cac cag cct gag ccg gag gag gag acc gag caa ccc aca ctg gactac 844 His Gln Pro Glu Pro Glu Glu Glu Thr Glu Gln Pro Thr Leu Asp Tyr220 225 230 235 aat gac cag atc gag agg gag gac tat gag gac ttt gag tacatt cgg 892 Asn Asp Gln Ile Glu Arg Glu Asp Tyr Glu Asp Phe Glu Tyr IleArg 240 245 250 cgc cag aag caa ccc agg cca ccc cca agc aga agg agg aggccc gag 940 Arg Gln Lys Gln Pro Arg Pro Pro Pro Ser Arg Arg Arg Arg ProGlu 255 260 265 cgg gtc tgg cca gag ccc cct gag gag aag gcc ccg gcc ccagcc ccg 988 Arg Val Trp Pro Glu Pro Pro Glu Glu Lys Ala Pro Ala Pro AlaPro 270 275 280 gag gag agg att gag cct cct gtg aag cct ctg ctg ccc ccgctg ccc 1036 Glu Glu Arg Ile Glu Pro Pro Val Lys Pro Leu Leu Pro Pro LeuPro 285 290 295 cct gac tat ggt gat ggt tac gtg atc ccc aac tac gat gacatg gac 1084 Pro Asp Tyr Gly Asp Gly Tyr Val Ile Pro Asn Tyr Asp Asp MetAsp 300 305 310 315 tat tac ttt ggg cct cct ccg ccc cag aag ccc gat gctgag cgc cag 1132 Tyr Tyr Phe Gly Pro Pro Pro Pro Gln Lys Pro Asp Ala GluArg Gln 320 325 330 acg gac gaa gag aag gag gag ctg aag aaa ccc aaa aaggag gac agc 1180 Thr Asp Glu Glu Lys Glu Glu Leu Lys Lys Pro Lys Lys GluAsp Ser 335 340 345 agc ccc aag gag gag acc gac aag tgg gca gtg gag aagggc aag gac 1228 Ser Pro Lys Glu Glu Thr Asp Lys Trp Ala Val Glu Lys GlyLys Asp 350 355 360 cac aaa gag ccc cga aag ggc gag gag ttg gag gag gagtgg acg cct 1276 His Lys Glu Pro Arg Lys Gly Glu Glu Leu Glu Glu Glu TrpThr Pro 365 370 375 acg gag aaa gtc aag tgt ccc ccc att ggg atg gag tcacac cgt att 1324 Thr Glu Lys Val Lys Cys Pro Pro Ile Gly Met Glu Ser HisArg Ile 380 385 390 395 gag gac aac cag atc cga gcc tcc tcc atg ctg cgccac ggc ctg ggg 1372 Glu Asp Asn Gln Ile Arg Ala Ser Ser Met Leu Arg HisGly Leu Gly 400 405 410 gca cag cgc ggc cgg ctc aac atg cag acc ggt gccact gag gac gac 1420 Ala Gln Arg Gly Arg Leu Asn Met Gln Thr Gly Ala ThrGlu Asp Asp 415 420 425 tac tat gat ggt gcg tgg tgt gcc gag gac gat gccagg acc cag tgg 1468 Tyr Tyr Asp Gly Ala Trp Cys Ala Glu Asp Asp Ala ArgThr Gln Trp 430 435 440 ata gag gtg gac acc agg agg act acc cgg ttc acaggc gtc atc acc 1516 Ile Glu Val Asp Thr Arg Arg Thr Thr Arg Phe Thr GlyVal Ile Thr 445 450 455 cag ggc aga gac tcc agc atc cat gac gat ttt gtgacc acc ttc ttc 1564 Gln Gly Arg Asp Ser Ser Ile His Asp Asp Phe Val ThrThr Phe Phe 460 465 470 475 gtg ggc ttc agc aat gac agc cag aca tgg gtgatg tac acc aac ggc 1612 Val Gly Phe Ser Asn Asp Ser Gln Thr Trp Val MetTyr Thr Asn Gly 480 485 490 tat gag gaa atg acc ttt cat ggg aac gtg gacaag gac aca ccc gtg 1660 Tyr Glu Glu Met Thr Phe His Gly Asn Val Asp LysAsp Thr Pro Val 495 500 505 ctg agt gag ctc cca gag ccg gtg gtg gct cgtttc atc cgc atc tac 1708 Leu Ser Glu Leu Pro Glu Pro Val Val Ala Arg PheIle Arg Ile Tyr 510 515 520 cca ctc acc tgg aat ggc agc ctg tgc atg cgcctg gag gtg ctg ggg 1756 Pro Leu Thr Trp Asn Gly Ser Leu Cys Met Arg LeuGlu Val Leu Gly 525 530 535 tgc tct gtg gcc cct gtc tac agc tac tac gcacag aat gag gtg gtg 1804 Cys Ser Val Ala Pro Val Tyr Ser Tyr Tyr Ala GlnAsn Glu Val Val 540 545 550 555 gcc acc gat gac ctg gat ttc cgg cac cacagc tac aag gac atg cgc 1852 Ala Thr Asp Asp Leu Asp Phe Arg His His SerTyr Lys Asp Met Arg 560 565 570 cag ctc atg aag gtg gtg aac gag gag tgcccc acc atc acc cgc act 1900 Gln Leu Met Lys Val Val Asn Glu Glu Cys ProThr Ile Thr Arg Thr 575 580 585 tac agc ctg ggc aag agc tca cga ggc ctcaag atc tat gcc atg gag 1948 Tyr Ser Leu Gly Lys Ser Ser Arg Gly Leu LysIle Tyr Ala Met Glu 590 595 600 atc tca gac aac cct ggg gag cat gaa ctgggg gag ccc gag ttc cgc 1996 Ile Ser Asp Asn Pro Gly Glu His Glu Leu GlyGlu Pro Glu Phe Arg 605 610 615 tac act gct ggg atc cat ggc aac gag gtgctg ggc cga gag ctg ttg 2044 Tyr Thr Ala Gly Ile His Gly Asn Glu Val LeuGly Arg Glu Leu Leu 620 625 630 635 ctg ctg ctc atg cag tac ctg tgc cgagag tac cgc gat ggg aac cca 2092 Leu Leu Leu Met Gln Tyr Leu Cys Arg GluTyr Arg Asp Gly Asn Pro 640 645 650 cgt gtg cgc agc ctg gtg cag gac acacgc atc cac ctg gtg ccc tca 2140 Arg Val Arg Ser Leu Val Gln Asp Thr ArgIle His Leu Val Pro Ser 655 660 665 ctg aac cct gat ggc tac gag gtg gcagcg cag atg ggc tca gag ttt 2188 Leu Asn Pro Asp Gly Tyr Glu Val Ala AlaGln Met Gly Ser Glu Phe 670 675 680 ggg aac tgg gcg ctg gga ctg tgg actgag gag ggc ttt gac atc ttt 2236 Gly Asn Trp Ala Leu Gly Leu Trp Thr GluGlu Gly Phe Asp Ile Phe 685 690 695 gaa gat ttc ccg gat ctc aac tct gtgctc tgg gga gct gag gag agg 2284 Glu Asp Phe Pro Asp Leu Asn Ser Val LeuTrp Gly Ala Glu Glu Arg 700 705 710 715 aaa tgg gtc ccc tac cgg gtc cccaac aat aac ttg ccc atc cct gaa 2332 Lys Trp Val Pro Tyr Arg Val Pro AsnAsn Asn Leu Pro Ile Pro Glu 720 725 730 cgc tac ctt tcg cca gat gcc acggta tcc acg gag gtc cgg gcc atc 2380 Arg Tyr Leu Ser Pro Asp Ala Thr ValSer Thr Glu Val Arg Ala Ile 735 740 745 att gcc tgg atg gag aag aac cccttc gtg ctg gga gca aat ctg aac 2428 Ile Ala Trp Met Glu Lys Asn Pro PheVal Leu Gly Ala Asn Leu Asn 750 755 760 ggc ggc gag cgg cta gta tcc tacccc tac gat atg gcc cgc acg cct 2476 Gly Gly Glu Arg Leu Val Ser Tyr ProTyr Asp Met Ala Arg Thr Pro 765 770 775 acc cag gag cag ctg ctg gcc gcagcc atg gca gca gcc cgg ggg gag 2524 Thr Gln Glu Gln Leu Leu Ala Ala AlaMet Ala Ala Ala Arg Gly Glu 780 785 790 795 gat gag gac gag gtc tcc gaggcc cag gag act cca gac cac gcc atc 2572 Asp Glu Asp Glu Val Ser Glu AlaGln Glu Thr Pro Asp His Ala Ile 800 805 810 ttc cgg tgg ctt gcc atc tccttc gcc tcc gca cac ctc acc ttg acc 2620 Phe Arg Trp Leu Ala Ile Ser PheAla Ser Ala His Leu Thr Leu Thr 815 820 825 gag ccc tac cgc gga ggc tgccaa gcc cag gac tac acc ggc ggc atg 2668 Glu Pro Tyr Arg Gly Gly Cys GlnAla Gln Asp Tyr Thr Gly Gly Met 830 835 840 ggc atc gtc aac ggg gcc aagtgg aac ccc cgg acc ggg act atc aat 2716 Gly Ile Val Asn Gly Ala Lys TrpAsn Pro Arg Thr Gly Thr Ile Asn 845 850 855 gac ttc agt tac ctg cat accaac tgc ctg gag ctc tcc ttc tac ctg 2764 Asp Phe Ser Tyr Leu His Thr AsnCys Leu Glu Leu Ser Phe Tyr Leu 860 865 870 875 ggc tgt gac aag ttc cctcat gag agt gag ctg ccc cgc gag tgg gag 2812 Gly Cys Asp Lys Phe Pro HisGlu Ser Glu Leu Pro Arg Glu Trp Glu 880 885 890 aac aac aag gag gcg ctgctc acc ttc atg gag cag gtg cac cgc ggc 2860 Asn Asn Lys Glu Ala Leu LeuThr Phe Met Glu Gln Val His Arg Gly 895 900 905 att aag ggg gtg gtg acggac gag caa ggc atc ccc att gcc aac gcc 2908 Ile Lys Gly Val Val Thr AspGlu Gln Gly Ile Pro Ile Ala Asn Ala 910 915 920 acc atc tct gtg agt ggcatt aat cac ggc gtg aag aca gcc agt ggt 2956 Thr Ile Ser Val Ser Gly IleAsn His Gly Val Lys Thr Ala Ser Gly 925 930 935 ggt gat tac tgg cga atcttg aac ccg ggt gag tac cgc gtg aca gcc 3004 Gly Asp Tyr Trp Arg Ile LeuAsn Pro Gly Glu Tyr Arg Val Thr Ala 940 945 950 955 cac gcg gag ggc tacacc ccg agc gcc aag acc tgc aat gtt gac tat 3052 His Ala Glu Gly Tyr ThrPro Ser Ala Lys Thr Cys Asn Val Asp Tyr 960 965 970 gac atc ggg gcc actcag tgc aac ttc atc ctg gct cgc tcc aac tgg 3100 Asp Ile Gly Ala Thr GlnCys Asn Phe Ile Leu Ala Arg Ser Asn Trp 975 980 985 aag cgc atc cgg gagatc atg gcc atg aac ggg aac cgg cct atc cca 3148 Lys Arg Ile Arg Glu IleMet Ala Met Asn Gly Asn Arg Pro Ile Pro 990 995 1000 cac ata gac cca tcgcgc cct atg acc ccc caa cag cga cgc ctg cag 3196 His Ile Asp Pro Ser ArgPro Met Thr Pro Gln Gln Arg Arg Leu Gln 1005 1010 1015 cag cga cgc ctacaa cac cgc ctg cgg ctt cgg gca cag atg cgg ctg 3244 Gln Arg Arg Leu GlnHis Arg Leu Arg Leu Arg Ala Gln Met Arg Leu 1020 1025 1030 1035 cgg cgcctc aac gcc acc acc acc cta ggc ccc cac act gtg cct ccc 3292 Arg Arg LeuAsn Ala Thr Thr Thr Leu Gly Pro His Thr Val Pro Pro 1040 1045 1050 acgctg ccc cct gcc cct gcc acc acc ctg agc act acc ata gag ccc 3340 Thr LeuPro Pro Ala Pro Ala Thr Thr Leu Ser Thr Thr Ile Glu Pro 1055 1060 1065tgg ggc ctc ata ccg cca acc acc gct ggc tgg gag gag tcg gag act 3388 TrpGly Leu Ile Pro Pro Thr Thr Ala Gly Trp Glu Glu Ser Glu Thr 1070 10751080 gag acc tac aca gag gtg gtg aca gag ttt ggg acc gag gtg gag ccc3436 Glu Thr Tyr Thr Glu Val Val Thr Glu Phe Gly Thr Glu Val Glu Pro1085 1090 1095 gag ttt ggg acc aag gtg gag ccc gag ttt gag acc cag ttggag cct 3484 Glu Phe Gly Thr Lys Val Glu Pro Glu Phe Glu Thr Gln Leu GluPro 1100 1105 1110 1115 gag ttc gag acc cag ctg gaa ccc gag ttt gag gaagag gag gag gag 3532 Glu Phe Glu Thr Gln Leu Glu Pro Glu Phe Glu Glu GluGlu Glu Glu 1120 1125 1130 gag aaa gag gag gag ata gcc act ggc cag gcattc ccc ttc aca aca 3580 Glu Lys Glu Glu Glu Ile Ala Thr Gly Gln Ala PhePro Phe Thr Thr 1135 1140 1145 gta gag acc tac aca gtg aac ttt ggg gacttc tgagatcagc gtcctaccaa 3633 Val Glu Thr Tyr Thr Val Asn Phe Gly AspPhe 1150 1155 gaccccagcc caactcaagc tacagcagca gcacttccca agcctgctgaccacagtcac 3693 atcacccatc agcacatgga aggcccctgg tatggacact gaaaggaagggctggtcctg 3753 cccctttgag ggggtgcaaa catgactggg acctaagagc cagaggctgtgtagaggctc 3813 ctgctccacc tgccagtctc gtaagagatg gggttgctgc agtgttggagtaggggcaga 3873 gggagggagc caaggtcact ccaataaaac aagctcatgg caaaaaaaaaaaaaaaaaaa 3933 aa 3935 2 1158 PRT Homo sapiens 2 Met Ala Ala Val ArgGly Ala Pro Leu Leu Ser Cys Leu Leu Ala Leu 1 5 10 15 Leu Ala Leu CysPro Gly Gly Arg Pro Gln Thr Val Leu Thr Asp Asp 20 25 30 Glu Ile Glu GluPhe Leu Glu Gly Phe Leu Ser Glu Leu Glu Pro Glu 35 40 45 Pro Arg Glu AspAsp Val Glu Ala Pro Pro Pro Pro Glu Pro Thr Pro 50 55 60 Arg Val Arg LysAla Gln Ala Gly Gly Lys Pro Gly Lys Arg Pro Gly 65 70 75 80 Thr Ala AlaGlu Val Pro Pro Glu Lys Thr Lys Asp Lys Gly Lys Lys 85 90 95 Gly Lys LysAsp Lys Gly Pro Lys Val Pro Lys Glu Ser Leu Glu Gly 100 105 110 Ser ProArg Pro Pro Lys Lys Gly Lys Glu Lys Pro Pro Lys Ala Thr 115 120 125 LysLys Pro Lys Glu Lys Pro Pro Lys Ala Thr Lys Lys Pro Lys Glu 130 135 140Glu Pro Pro Lys Ala Thr Lys Lys Pro Lys Glu Lys Pro Pro Lys Ala 145 150155 160 Thr Lys Lys Pro Pro Ser Gly Lys Arg Pro Pro Ile Leu Ala Pro Ser165 170 175 Glu Thr Leu Glu Trp Pro Leu Pro Pro Pro Pro Ser Pro Gly ProGlu 180 185 190 Glu Leu Pro Gln Glu Gly Gly Ala Pro Leu Ser Asn Asn TrpGln Asn 195 200 205 Pro Gly Glu Glu Thr His Val Glu Ala Gln Glu His GlnPro Glu Pro 210 215 220 Glu Glu Glu Thr Glu Gln Pro Thr Leu Asp Tyr AsnAsp Gln Ile Glu 225 230 235 240 Arg Glu Asp Tyr Glu Asp Phe Glu Tyr IleArg Arg Gln Lys Gln Pro 245 250 255 Arg Pro Pro Pro Ser Arg Arg Arg ArgPro Glu Arg Val Trp Pro Glu 260 265 270 Pro Pro Glu Glu Lys Ala Pro AlaPro Ala Pro Glu Glu Arg Ile Glu 275 280 285 Pro Pro Val Lys Pro Leu LeuPro Pro Leu Pro Pro Asp Tyr Gly Asp 290 295 300 Gly Tyr Val Ile Pro AsnTyr Asp Asp Met Asp Tyr Tyr Phe Gly Pro 305 310 315 320 Pro Pro Pro GlnLys Pro Asp Ala Glu Arg Gln Thr Asp Glu Glu Lys 325 330 335 Glu Glu LeuLys Lys Pro Lys Lys Glu Asp Ser Ser Pro Lys Glu Glu 340 345 350 Thr AspLys Trp Ala Val Glu Lys Gly Lys Asp His Lys Glu Pro Arg 355 360 365 LysGly Glu Glu Leu Glu Glu Glu Trp Thr Pro Thr Glu Lys Val Lys 370 375 380Cys Pro Pro Ile Gly Met Glu Ser His Arg Ile Glu Asp Asn Gln Ile 385 390395 400 Arg Ala Ser Ser Met Leu Arg His Gly Leu Gly Ala Gln Arg Gly Arg405 410 415 Leu Asn Met Gln Thr Gly Ala Thr Glu Asp Asp Tyr Tyr Asp GlyAla 420 425 430 Trp Cys Ala Glu Asp Asp Ala Arg Thr Gln Trp Ile Glu ValAsp Thr 435 440 445 Arg Arg Thr Thr Arg Phe Thr Gly Val Ile Thr Gln GlyArg Asp Ser 450 455 460 Ser Ile His Asp Asp Phe Val Thr Thr Phe Phe ValGly Phe Ser Asn 465 470 475 480 Asp Ser Gln Thr Trp Val Met Tyr Thr AsnGly Tyr Glu Glu Met Thr 485 490 495 Phe His Gly Asn Val Asp Lys Asp ThrPro Val Leu Ser Glu Leu Pro 500 505 510 Glu Pro Val Val Ala Arg Phe IleArg Ile Tyr Pro Leu Thr Trp Asn 515 520 525 Gly Ser Leu Cys Met Arg LeuGlu Val Leu Gly Cys Ser Val Ala Pro 530 535 540 Val Tyr Ser Tyr Tyr AlaGln Asn Glu Val Val Ala Thr Asp Asp Leu 545 550 555 560 Asp Phe Arg HisHis Ser Tyr Lys Asp Met Arg Gln Leu Met Lys Val 565 570 575 Val Asn GluGlu Cys Pro Thr Ile Thr Arg Thr Tyr Ser Leu Gly Lys 580 585 590 Ser SerArg Gly Leu Lys Ile Tyr Ala Met Glu Ile Ser Asp Asn Pro 595 600 605 GlyGlu His Glu Leu Gly Glu Pro Glu Phe Arg Tyr Thr Ala Gly Ile 610 615 620His Gly Asn Glu Val Leu Gly Arg Glu Leu Leu Leu Leu Leu Met Gln 625 630635 640 Tyr Leu Cys Arg Glu Tyr Arg Asp Gly Asn Pro Arg Val Arg Ser Leu645 650 655 Val Gln Asp Thr Arg Ile His Leu Val Pro Ser Leu Asn Pro AspGly 660 665 670 Tyr Glu Val Ala Ala Gln Met Gly Ser Glu Phe Gly Asn TrpAla Leu 675 680 685 Gly Leu Trp Thr Glu Glu Gly Phe Asp Ile Phe Glu AspPhe Pro Asp 690 695 700 Leu Asn Ser Val Leu Trp Gly Ala Glu Glu Arg LysTrp Val Pro Tyr 705 710 715 720 Arg Val Pro Asn Asn Asn Leu Pro Ile ProGlu Arg Tyr Leu Ser Pro 725 730 735 Asp Ala Thr Val Ser Thr Glu Val ArgAla Ile Ile Ala Trp Met Glu 740 745 750 Lys Asn Pro Phe Val Leu Gly AlaAsn Leu Asn Gly Gly Glu Arg Leu 755 760 765 Val Ser Tyr Pro Tyr Asp MetAla Arg Thr Pro Thr Gln Glu Gln Leu 770 775 780 Leu Ala Ala Ala Met AlaAla Ala Arg Gly Glu Asp Glu Asp Glu Val 785 790 795 800 Ser Glu Ala GlnGlu Thr Pro Asp His Ala Ile Phe Arg Trp Leu Ala 805 810 815 Ile Ser PheAla Ser Ala His Leu Thr Leu Thr Glu Pro Tyr Arg Gly 820 825 830 Gly CysGln Ala Gln Asp Tyr Thr Gly Gly Met Gly Ile Val Asn Gly 835 840 845 AlaLys Trp Asn Pro Arg Thr Gly Thr Ile Asn Asp Phe Ser Tyr Leu 850 855 860His Thr Asn Cys Leu Glu Leu Ser Phe Tyr Leu Gly Cys Asp Lys Phe 865 870875 880 Pro His Glu Ser Glu Leu Pro Arg Glu Trp Glu Asn Asn Lys Glu Ala885 890 895 Leu Leu Thr Phe Met Glu Gln Val His Arg Gly Ile Lys Gly ValVal 900 905 910 Thr Asp Glu Gln Gly Ile Pro Ile Ala Asn Ala Thr Ile SerVal Ser 915 920 925 Gly Ile Asn His Gly Val Lys Thr Ala Ser Gly Gly AspTyr Trp Arg 930 935 940 Ile Leu Asn Pro Gly Glu Tyr Arg Val Thr Ala HisAla Glu Gly Tyr 945 950 955 960 Thr Pro Ser Ala Lys Thr Cys Asn Val AspTyr Asp Ile Gly Ala Thr 965 970 975 Gln Cys Asn Phe Ile Leu Ala Arg SerAsn Trp Lys Arg Ile Arg Glu 980 985 990 Ile Met Ala Met Asn Gly Asn ArgPro Ile Pro His Ile Asp Pro Ser 995 1000 1005 Arg Pro Met Thr Pro GlnGln Arg Arg Leu Gln Gln Arg Arg Leu Gln 1010 1015 1020 His Arg Leu ArgLeu Arg Ala Gln Met Arg Leu Arg Arg Leu Asn Ala 1025 1030 1035 1040 ThrThr Thr Leu Gly Pro His Thr Val Pro Pro Thr Leu Pro Pro Ala 1045 10501055 Pro Ala Thr Thr Leu Ser Thr Thr Ile Glu Pro Trp Gly Leu Ile Pro1060 1065 1070 Pro Thr Thr Ala Gly Trp Glu Glu Ser Glu Thr Glu Thr TyrThr Glu 1075 1080 1085 Val Val Thr Glu Phe Gly Thr Glu Val Glu Pro GluPhe Gly Thr Lys 1090 1095 1100 Val Glu Pro Glu Phe Glu Thr Gln Leu GluPro Glu Phe Glu Thr Gln 1105 1110 1115 1120 Leu Glu Pro Glu Phe Glu GluGlu Glu Glu Glu Glu Lys Glu Glu Glu 1125 1130 1135 Ile Ala Thr Gly GlnAla Phe Pro Phe Thr Thr Val Glu Thr Tyr Thr 1140 1145 1150 Val Asn PheGly Asp Phe 1155 3 2743 DNA Mus musculus 3 aagcttagtc tccctctctcctggctcctc tcctggggct tccctatgga ggtagcactt 60 acagaagatg cttgttccaaaccttcaggg gtacaaacta cacagatata ctgaaggaca 120 ggaggctggg gcctccccccacccccaaca gccactgttc tctcaggagc tctgcttctg 180 ctctgcagca ttgaaaacaaaactgaagga caccttcctt ctctcaggcc agcccagtgc 240 tgttgtgtga tccctcgggaagactctaac gcattcacag ggacaacagg agttgggagg 300 gagaggagtt acagaactttccagcaggac ctcaggagaa cgcctggaca cggacaggaa 360 cccccaaccc ctcagggacccccttggacc ctttgagtgc tcctgatcat ggaagccacc 420 agcctcccga ttcctcagctgtggccttgg cagtgccctc tggacatttg acttaaacgc 480 tatgctcttc agcagagtggagagctctcc tcacaggctc tggcttctgg ttgtcctctt 540 gccccagcgc tgtgggcccaggttagaaag acttcctgag gacaggctcc ctcaggagga 600 tccccagcgt acgactgtgctcccacgcac ctttccggat tttctgtgtg gaggcctcaa 660 cccctcaggc ctcctgggccagctcctctg ctcgaattcc tgtccgtgac tcattgaggc 720 tcaggaaaag gctttctagaccttaggttt ctttgttttc catttttgaa atggcttctg 780 ttttccctgg cagagaatatccaacccaaa ttcagtccaa gtatgaccca tgcctaggga 840 agtgacatcc atgtcccctcatgcaccctg tggcataccc agcatgacac actggaccag 900 actgggggca cggaagccaattcccagaac tgactttgag cacaatgatt cagagggtga 960 ccatgagtga gacttgctttactcttgctc tgcgaccagg ttgaagtctc tcatggggag 1020 gcctagctgt gagaggattgtcctgggatg ggggaagggg gagcaaagtg gatgaggacc 1080 aacagcctgt gggatgcaagggctgatcgt gtgtgctagg cacagcacaa agtggtccat 1140 ttagccgggc agtggtggtgcacaccttta atcccagcac ttgggaggca gcagcaggtg 1200 ggtttctgag ttcgaggccagcctggtcta cagagcaagt tccaggacag ccagagctac 1260 acagagaaac tctgtctcaaaaaaatcgaa taaaccagaa aggtggtcca tttaatatgc 1320 gtatagtaag ttgtggacacgggagttccc ctgctgagtc agacagctag gagggctaag 1380 atgggttaga ccctccccccccccacacac acacacacac actcacacac acatcagttc 1440 ttggcatagt ctccatgcttcctcaaggag agccagaaag gagactgccg ggaggagctt 1500 gcctactccc tgagagcagtgggttacaga gcccagtgcc cgaaaatttc ccctttttct 1560 ccctgctcat gctggacagagagggtgagg gtgagggtga aagactgagg aggtggcatc 1620 gtgttggtgt ttcttgacctgctttttctt ttttctcttc cagctgagat gtaaactttc 1680 ccatgtcaat catctgggggtcgctattct tttttatcag agtgcctccc caccttggtt 1740 gaaagctgcc tgccactaccctggacctat ggctgctaca agcccacgtt cacatcttta 1800 atccttcatg ggtaaatgctctggcattcc tgggcttagc tatgatggcc attatgagcc 1860 agccaacgtt tgtattctagaagccatagc tgaagctgtt gtaaacaatt tgttgtttta 1920 accgcttctg gtcagaggaaggagagaata gctattactc cacattggga cctgagccct 1980 gagctctgaa gtggggctcctatctccata aggacagcag cttgctgaga acagcttttc 2040 acagccttcc tcgcaaaaattggctccaaa gacctgggat gttggtgata actggacaaa 2100 ggtgacacct gtgcaagcacacagcaggtg acactttgaa gagctaacct ccagaaagtg 2160 gaaaggaggt gatcgccagtaccctcgagg gccctactcc ctccctcccc tagcaatctc 2220 cctgggctca gagcaaagggcacagcgggt tagagcacag gtctccttag actccgcaca 2280 ctcccttccc cataactgttgcattctttt ctcccaggcc ttcctccccg ctaggcgccc 2340 tgcacccaga ccctctaaactggcgcgtga cgctgctatt agtctgggct ccgtgctgtc 2400 cgcctccctc ccccgcagcccccggtccaa ggccggctcc tcctcctccc cctccggaaa 2460 cccgaagccc ccgccccggccaggccgtcg caagcgctct ggagggcggt ccgcgtgaga 2520 gccagccacg cggggcaggagcgcccagtt gctgccggag ctgggcccgc cagaacctct 2580 cctggagccc cttgctctccttgaatctcc ctttcccacc gctttctgga tacccttgac 2640 gcccacgttc ctcgcgccctttcccgcccc tacgcggggc gctgcccctg ccacccaagt 2700 ccctgctcaa gcccgcccggtcccgcgcgt gcccagagcc atg 2743 4 17 DNA Mus musculus 4 atctggttgtcctcaat 17 5 17 DNA Mus musculus 5 tgactccatc ccaatag 17 6 26 DNA Musmusculus 6 cggaattcag tccctgctca agcccg 26 7 28 DNA Mus musculus 7cgaagcttga agtccccaaa gttcactg 28 8 1128 PRT Mus musculus 8 Met Ala ProVal Arg Thr Ala Ser Leu Leu Cys Gly Leu Leu Ala Leu 1 5 10 15 Leu ThrLeu Cys Pro Glu Gly Asn Pro Gln Thr Val Leu Thr Asp Asp 20 25 30 Glu IleGlu Glu Phe Leu Glu Gly Phe Leu Ser Glu Leu Glu Thr Gln 35 40 45 Ser ProPro Arg Glu Asp Asp Val Glu Val Gln Pro Leu Pro Glu Pro 50 55 60 Thr GlnArg Pro Arg Lys Ser Lys Ala Gly Gly Lys Gln Arg Ala Asp 65 70 75 80 ValGlu Val Pro Pro Glu Lys Asn Lys Asp Lys Glu Lys Lys Gly Lys 85 90 95 LysAsp Lys Gly Pro Lys Ala Thr Lys Pro Leu Glu Gly Ser Thr Arg 100 105 110Pro Thr Lys Lys Pro Lys Glu Lys Pro Pro Lys Ala Thr Lys Lys Pro 115 120125 Lys Glu Lys Pro Pro Lys Ala Thr Lys Lys Pro Lys Glu Lys Pro Pro 130135 140 Lys Ala Thr Lys Lys Pro Lys Glu Lys Pro Pro Lys Ala Thr Lys Arg145 150 155 160 Pro Ser Ala Gly Lys Lys Phe Ser Thr Val Ala Pro Leu GluThr Leu 165 170 175 Asp Arg Leu Leu Pro Ser Pro Ser Asn Pro Ser Ala GlnGlu Leu Pro 180 185 190 Gln Lys Arg Asp Thr Pro Phe Pro Asn Ala Trp GlnGly Gln Gly Glu 195 200 205 Glu Thr Gln Val Glu Ala Lys Gln Pro Arg ProGlu Pro Glu Glu Glu 210 215 220 Thr Glu Met Pro Thr Leu Asp Tyr Asn AspGln Ile Glu Lys Glu Asp 225 230 235 240 Tyr Glu Asp Phe Glu Tyr Ile ArgArg Gln Lys Gln Pro Arg Pro Thr 245 250 255 Pro Ser Arg Arg Arg Leu TrpPro Glu Arg Pro Glu Glu Lys Thr Glu 260 265 270 Glu Pro Glu Glu Arg LysGlu Val Glu Pro Pro Leu Lys Pro Leu Leu 275 280 285 Pro Pro Asp Tyr GlyAsp Ser Tyr Val Ile Pro Asn Tyr Asp Asp Leu 290 295 300 Asp Tyr Tyr PhePro His Pro Pro Pro Gln Lys Pro Asp Val Gly Gln 305 310 315 320 Glu ValAsp Glu Glu Lys Glu Glu Met Lys Lys Pro Lys Lys Glu Gly 325 330 335 SerSer Pro Lys Glu Asp Thr Glu Asp Lys Trp Thr Val Glu Lys Asn 340 345 350Lys Asp His Lys Gly Pro Arg Lys Gly Glu Glu Leu Glu Glu Glu Trp 355 360365 Ala Pro Val Glu Lys Ile Lys Cys Pro Pro Ile Gly Met Glu Ser His 370375 380 Arg Ile Glu Asp Asn Gln Ile Arg Ala Ser Ser Met Leu Arg His Gly385 390 395 400 Leu Gly Ala Gln Arg Gly Arg Leu Asn Met Gln Ala Gly AlaAsn Glu 405 410 415 Asp Asp Tyr Tyr Asp Gly Ala Trp Cys Ala Glu Asp GluSer Gln Thr 420 425 430 Gln Trp Ile Glu Val Asp Thr Arg Arg Thr Thr ArgPhe Thr Gly Val 435 440 445 Ile Thr Gln Gly Arg Asp Ser Ser Ile His AspAsp Phe Val Thr Thr 450 455 460 Phe Phe Val Gly Phe Ser Asn Asp Ser GlnThr Trp Val Met Tyr Thr 465 470 475 480 Asn Gly Tyr Glu Glu Met Thr PheTyr Gly Asn Val Asp Lys Asp Thr 485 490 495 Pro Val Leu Ser Glu Leu ProGlu Pro Val Val Ala Arg Phe Ile Arg 500 505 510 Ile Tyr Pro Leu Thr TrpAsn Gly Ser Leu Cys Met Arg Leu Glu Val 515 520 525 Leu Gly Cys Pro ValThr Pro Val Tyr Ser Tyr Tyr Ala Gln Asn Glu 530 535 540 Val Val Thr ThrAsp Ser Leu Asp Phe Arg His His Ser Tyr Lys Asp 545 550 555 560 Met ArgGln Leu Met Lys Ala Val Asn Glu Glu Cys Pro Thr Ile Thr 565 570 575 ArgThr Tyr Ser Leu Gly Lys Ser Ser Arg Gly Leu Lys Ile Tyr Ala 580 585 590Met Glu Ile Ser Asp Asn Pro Gly Asp His Glu Leu Gly Glu Pro Glu 595 600605 Phe Arg Tyr Thr Ala Gly Ile His Gly Asn Glu Val Leu Gly Arg Glu 610615 620 Leu Leu Leu Leu Leu Met Gln Tyr Leu Cys Gln Glu Tyr Arg Asp Gly625 630 635 640 Asn Pro Arg Val Arg Asn Leu Val Gln Asp Thr Arg Ile HisLeu Val 645 650 655 Pro Ser Leu Asn Pro Asp Gly Tyr Glu Val Ala Ala GlnMet Gly Ser 660 665 670 Glu Phe Gly Asn Trp Ala Leu Gly Leu Trp Thr GluGlu Gly Phe Asp 675 680 685 Ile Phe Glu Asp Phe Pro Asp Leu Asn Ser ValLeu Trp Ala Ala Glu 690 695 700 Glu Lys Lys Trp Val Pro Tyr Arg Val ProAsn Asn Asn Leu Pro Ile 705 710 715 720 Pro Glu Arg Tyr Leu Ser Pro AspAla Thr Val Ser Thr Glu Val Arg 725 730 735 Ala Ile Ile Ser Trp Met GluLys Asn Pro Phe Val Leu Gly Ala Asn 740 745 750 Leu Asn Gly Gly Glu ArgLeu Val Ser Tyr Pro Tyr Asp Met Ala Arg 755 760 765 Thr Pro Ser Gln GluGln Leu Leu Ala Glu Ala Leu Ala Ala Ala Arg 770 775 780 Gly Glu Asp AspAsp Gly Val Ser Glu Ala Gln Glu Thr Pro Asp His 785 790 795 800 Ala IlePhe Arg Trp Leu Ala Ile Ser Phe Ala Ser Ala His Leu Thr 805 810 815 MetThr Glu Pro Tyr Arg Gly Gly Cys Gln Ala Gln Asp Tyr Thr Ser 820 825 830Gly Met Gly Ile Val Asn Gly Ala Lys Trp Asn Pro Arg Ser Gly Thr 835 840845 Phe Asn Arg Phe Ser Tyr Leu His Thr Asn Cys Leu Glu Leu Ser Val 850855 860 Tyr Leu Gly Cys Asp Lys Phe Pro His Glu Ser Glu Leu Pro Arg Glu865 870 875 880 Trp Glu Asn Asn Lys Glu Ala Leu Leu Thr Phe Met Glu GlnVal His 885 890 895 Arg Gly Ile Lys Gly Val Val Thr Asp Glu Gln Gly IlePro Ile Ala 900 905 910 Asn Ala Thr Ile Ser Val Ser Gly Ile Asn His GlyVal Lys Thr Ala 915 920 925 Ser Gly Gly Asp Tyr Trp Arg Ile Leu Asn ProGly Glu Tyr Arg Val 930 935 940 Thr Ala His Ala Glu Gly Tyr Thr Ser SerAla Lys Ile Cys Asn Val 945 950 955 960 Asp Tyr Asp Ile Gly Ala Thr GlnCys Asn Phe Ile Leu Ala Arg Ser 965 970 975 Asn Trp Lys Arg Ile Arg GluIle Leu Ala Met Asn Gly Asn Arg Pro 980 985 990 Ile Leu Gly Val Asp ProSer Arg Pro Met Thr Pro Gln Gln Arg Arg 995 1000 1005 Met Gln Gln ArgArg Leu Gln Tyr Arg Leu Arg Met Arg Glu Gln Met 1010 1015 1020 Arg LeuArg Arg Leu Asn Ser Thr Ala Gly Pro Ala Thr Ser Pro Thr 1025 1030 10351040 Pro Ala Leu Met Pro Pro Pro Ser Pro Thr Pro Ala Ile Thr Leu Arg1045 1050 1055 Pro Trp Glu Val Leu Pro Thr Thr Thr Ala Gly Trp Glu GluSer Glu 1060 1065 1070 Thr Glu Thr Tyr Thr Glu Val Val Thr Glu Phe GluThr Glu Tyr Gly 1075 1080 1085 Thr Asp Leu Glu Val Glu Glu Ile Glu GluGlu Glu Glu Glu Glu Glu 1090 1095 1100 Glu Glu Met Asp Thr Gly Leu ThrPhe Pro Leu Thr Thr Val Glu Thr 1105 1110 1115 1120 Tyr Thr Val Asn PheGly Asp Phe 1125

1. An isolated nucleic acid molecule encoding a human aorticcarboxypeptidase like polypeptide (ACLP).
 2. The nucleic acid moleculeof claim 1, wherein said nucleic acid molecule comprises the nucleotides140-3613, inclusive, of SEQ ID NO: 1 or a degenerate variant thereof. 3.A nucleic acid molecule comprising a nucleotide sequence encoding apolypeptide having a sequence that is at least 87% identical to thesequence of SEQ ID NO:
 1. 4. An isolated nucleic acid moleculecomprising a strand which hybridizes at high stringency to a DNA havingthe sequence of SEQ ID NO: 1, or the complement thereof.
 5. The nucleicacid molecule of claim 4, wherein said molecule is at least 5nucleotides but less than 50 nucleotides in length.
 6. The nucleic acidmolecule of claim 4, wherein said molecule comprises nucleotides140-3613, inclusive, of SEQ ID NO:
 1. 7. The nucleic acid molecule ofclaim 4, wherein said molecule spans a gastroschisis-associated mutationin an ACLP gene.
 8. The nucleic acid molecule of claim 1, wherein saidnucleic acid molecule is operably linked to a regulatory sequence forexpression of said polypeptide, said regulatory sequence comprising apromoter.
 9. A substantially pure human ACLP.
 10. The polypeptide ofclaim 9, said polypeptide comprising an amino acid sequence that is atleast 87% identical to the amino acid sequence of SEQ ID NO:
 2. 11. Thepolypeptide of claim 10, said polypeptide comprising an amino acidsequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO:
 2. 12. The polypeptide of claim 11, wherein said polypeptidecomprises the amino acid sequence of SEQ ID NO:
 2. 13. The method ofclaim 9, wherein said ACLP comprises amino acids 117-164 of SEQ ID NO:2.
 14. The method of claim 9, wherein said ACLP comprises amino acids385-540 of SEQ ID NO:
 2. 15. The method of claim 9, wherein said ACLPcomprises amino acids 562-969 of SEQ ID NO:
 2. 16. A cell comprising thenucleic acid molecule of claim
 1. 17. A method of making an ACLP,comprising (a) providing the cell of claim 16, and (b)culturing it underconditions permitting expression of said nucleic acid molecule.
 18. Asubstantially pure DNA comprising an ACLP promoter/enhancer sequence.19. The DNA of claim 18, wherein said promoter/enhancer sequencecomprises SEQ ID NO:3.
 20. The DNA of claim 18, wherein saidpromoter/enhancer sequence is operably linked to a polypeptide-encodingsequence and regulates transcription of said polypeptide-encodingsequence.
 21. The DNA of claim 18, wherein said promoter/enhancersequence regulates developmental stage-specific expression of saidpolypeptide-encoding sequence.
 22. The DNA of claim 21, wherein saidpromoter/enhancer sequence directs transcription of saidpolypeptide-encoding sequence in vascular cells of the gastrointestinaltract.
 23. The DNA of claim 20, wherein said polypeptide-encodingsequence does not encode an ACLP.
 24. The DNA of claim 20 wherein saidpolypeptide-encoding sequence encodes human ACLP.
 25. A vectorcomprising the DNA of claim
 18. 26. A method of detecting agastroschisis-associated genetic alteration comprising (a) providing asample of DNA from a patient or fetus; and (b) determining whether saidDNA comprises a mutation in a gene encoding an ACLP, said mutation beingan indication that said patient or fetus has a gastroschisis-associatedgenetic alteration.
 27. The method of claim 26, wherein said sample isobtained by amniocentesis or chorionic villi sampling.
 28. The method ofclaim 26, wherein said method includes the step of subjecting saidsample to polymerase chain reaction (PCR), using a forward PCR primercomplementary to a portion of the antisense strand of said gene, saidportion being within (a) a first intron of said gene, or (b) the 5′untranslated region adjacent to the start codon of said gene; and areverse PCR primer complementary to a fragment of the sense strand ofsaid gene, said fragment being within (a) a second intron of said gene,or (b) the 3′ untranslated region adjacent to the termination codon ofsaid gene.
 29. An antibody that selectively binds to an ACLP.
 30. Theantibody of claim 29, wherein said antibody is a monoclonal antibody.31. A method of diagnosing gastroschisis or a predisposition theretocomprising a) providing a tissue sample from a patient or fetus; and b)measuring expression of an ACLP gene in said tissue sample, wherein areduction in said expression in said tissue sample compared to that in anormal control tissue sample indicates that said patient or fetus has oris predisposed to developing gastroschisis.
 32. The method of claim 31,wherein said expression is measured by contacting said tissue samplewith an ACLP-specific antibody.
 33. A method of treating or preventingthe development of gastroschisis, comprising (a) identifying a patientwith or at risk of developing gastroschisis; and (b) introducing intocells of said patient an isolated nucleic acid encoding an ACLP.
 34. Themethod of claim 33, wherein the isolated nucleic acid comprises thenucleotide sequence of 140 to 3613 of SEQ ID NO:1.
 35. A method oftreating or preventing the development of gastroschisis, comprising (a)identifying a patient with or at risk of developing gastroschisis; and(b) introducing an ACLP into cells of said patient.
 36. The method ofclaim 35, wherein said ACLP comprises the amino acid sequence of SEQ IDNO:2.
 37. A genetically-altered non-human mammal all diploid cells ofwhich contain a mutation in an endogenous gene encoding an ACLP.
 38. Themammal of claim 37, wherein said mutation is a null mutation.
 39. Themammal of claim 37, wherein the mammal is a rodent.
 40. The mammal ofclaim 39, wherein the rodent is a mouse.